HK1171083B - Nucleic acid analysis - Google Patents

Description

nucleic acid analysis

Reference to related applications

This application claims priority to U.S. provisional applications 61,226,025 and 61,226,106 filed on 7, 16, 2009, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the general field of nucleic acid analysis in human or other animal subjects, particularly the acquisition and analysis of high quality nucleic acids from biological samples, particularly microvesicles.

Background

Small microvesicles shed by cells are called "exosomes" (Thery et al, 2002). Exosomes have been reported to be about 30-100nm in diameter, and many different cell types shed exosomes in both normal and pathological states (Thery et al, 2002). Exosomes are typically formed by inward invagination and pinching off of late endosomal membranes. This results in the formation of multivesicular bodies (MVB) filled with small lipid bilayer vesicles (-40-100 nm in diameter), each containing a cytoplasmic sample of the blast (Stoorvogel et al, 2002). The fusion of the MVB to the cell membrane results in the release of these exosomes from the cell and delivery into the blood, urine or other body fluids.

Another class of cell-derived vesicles is known as "shedding microvesicles" (Cocucci et al, 2009). These microvesicles, which are formed by direct budding of the cytoplasmic membrane, are more heterogeneous in size than exosomes and, like exosomes, also contain cytoplasmic samples of the mother cells. Exosomes and shedding microvesicles can be co-isolated using ultracentrifugation and ultrafiltration separation techniques and are therefore collectively referred to as microvesicles.

Recent studies have revealed that nucleic acids within the microvesicles have a role as biomarkers. For example, Skog et al describe, among other things, the use of nucleic acids extracted from microvesicles in the serum of GBM patients in medical diagnosis, prognosis and treatment evaluation (Skog et al, 2008). The use of nucleic acids extracted from microvesicles is considered to potentially circumvent the need for biopsy, emphasizing the enormous diagnostic potential of microvesicle biology (Skog et al, 2008).

In the research and development of nucleic acid biomarkers and industrial applications, it is desirable to extract high quality nucleic acids from biological samples in a consistent and reliable manner. The present invention provides compositions of high quality nucleic acid extracts from microvesicles and other biological samples, methods of making such extracts, and methods of using these high quality nucleic acids in various applications.

Disclosure of Invention

In one aspect, the invention is a novel nucleic acid extract from one or more microvesicles isolated from a eukaryotic sample, wherein 18s rrna and 28s rrna are detectable in the extract. Preferably, the quantitative ratio of 18SrRNA to 28SrRNA detectable in the novel extract is in the range of about 1: 1 to about 1: 2, preferably about 1: 2. The biological samples from which the novel extract can be obtained include, inter alia, any body fluid, preferably urine, serum or plasma, preferably from a mammal, in particular a human. For a body fluid sample with a protein concentration below 10mg/ml, such as urine, the novel nucleic acid extract may further comprise a nucleic acid extract with an RNA integrity (in all cases, as obtained on an agilent bioanalyzer or equivalent) of greater than or equal to 5, and/or may further comprise a nucleic acid yield from a 20ml biological sample of greater than or equal to 50 pg/ml. Similarly, for a body fluid sample having a protein concentration greater than 10mg/ml, such as serum or plasma, the novel nucleic acid extract may further comprise an RNA integrity of greater than or equal to 3, and/or may further comprise a nucleic acid yield of greater than or equal to 50pg/ml from a 1ml biological sample.

In another aspect, the invention is a novel nucleic acid profile from one or more microvesicles isolated from a eukaryotic sample, wherein 18s rrna and 28s rrna can be detected in the profile. Preferably, the quantitative ratio of 18SrRNA to 28SrRNA detectable in the new profile is in the range of about 1: 1 to about 1: 2, and preferably about 1: 2. Biological samples from which the novel profile can be obtained include, inter alia, any body fluid, preferably urine, serum or plasma, preferably from a mammal, in particular a human. For a body fluid sample with a protein concentration below 10mg/ml, such as urine, the new profile may further comprise an RNA integrity of greater than or equal to 5, and/or may further comprise a nucleic acid yield from 20ml of the biological sample of greater than or equal to 50 pg/ml. Similarly, for a body fluid sample with a protein concentration greater than 10mg/ml, such as serum or plasma, the novel profile may further comprise an RNA integrity of greater than or equal to 3, and/or may further comprise a nucleic acid yield from 1ml of the biological sample of greater than or equal to 50 pg/ml.

In yet another aspect, the invention is a method of assessing the quality of a nucleic acid extract from microvesicles isolated from a eukaryotic sample, comprising the steps of: (a) extracting RNA from the microvesicles; and (b) measuring the mass of RNA by determining the amount of 18SrRNA and 28SrRNA in the extract. Preferably, the quantitative ratio of 18SrRNA to 28SrRNA determined in the novel method is in the range of about 1: 1 to about 1: 2, preferably about 1: 2. The biological samples on which the novel method can be carried out include, inter alia, any body fluid, preferably urine, serum or plasma, preferably from a mammal, in particular a human. For body fluid samples with a protein concentration below 10mg/ml, such as urine, the novel method may further produce a nucleic acid extract with an RNA integrity greater than or equal to 5, and/or may further produce a nucleic acid yield from 20ml of biological sample greater than or equal to 50 pg/ml. Similarly, for a bodily fluid sample with a protein concentration greater than 10mg/ml, such as serum or plasma, the novel method can further produce a nucleic acid extract with an RNA integrity greater than or equal to 3, and/or can further produce a nucleic acid yield greater than or equal to 50pg/ml from a 1ml biological sample.

In a further aspect, the invention is a method for obtaining nucleic acids from a biological sample comprising the steps of: (a) obtaining a biological sample; (b) performing an extraction-facilitating operation on the biological sample; and (c) extracting nucleic acids from the biological sample. The extraction-promoting operation consists of the following steps: (a) adding one or more enhancers to the biological sample; or (b) performing one or more facilitating steps prior to extracting the nucleic acid; or (c) a combination of the addition of an accelerator and the accelerating step. The accelerator may include: (i) an RNase inhibitor; (ii) a protease; (iii) a reducing agent; (iv) bait substrates (decoysubstrates), such as synthetic RNA; (v) a soluble receptor; (vi) a small interfering RNA; (vii) RNA binding molecules, such as anti-RNA antibodies, chaperones, or rnase inhibitory proteins; (ix) RNase denaturing substances such as high osmotic pressure solutions or detergents. The extraction facilitating step may include: (x) Washing; (xi) Performing coarse and fine sorting on the RNA enzyme in the sample; (xii) The rnase is denatured by physical changes, such as by lowering the temperature, or performing freeze/thaw cycles. The novel method may be performed on a biological sample comprising, inter alia, any body fluid, preferably urine, serum or plasma, preferably from a mammal, in particular a human. In one embodiment, a derivative is obtained from the biological sample and an extraction-facilitating operation is performed on the derivative prior to extracting the nucleic acid. Preferably, the derivative is a microvesicle fraction derived from a biological sample. In one embodiment, the microvesicle fraction is obtained by filtration concentration techniques, but other known separation techniques may also be utilized. In a further aspect of the method of the invention, the derivative may be treated with a ribonuclease, a deoxyribonuclease, or a combination thereof, prior to being subjected to the facilitated extraction operation. In some aspects, the extraction enhancement operation comprises adding an rnase inhibitor to the biological sample, or to the derivative, prior to extracting nucleic acids; preferably the RNase inhibitor has a concentration of more than 0.027AU (1X) at a sample of 1. mu.l or more; alternatively, the sample has a concentration of greater than or equal to 0.135AU (5X) at or above 1 μ l; alternatively, a concentration of greater than or equal to 0.27AU (10X) at 1 μ l or greater of the sample; alternatively, the sample has a concentration of greater than or equal to 0.675AU (25X) at or greater than 1 μ l; alternatively, the sample has a concentration of 1.35AU (50X) or more at 1. mu.l or more, wherein the 1X protease concentration relates to an enzymatic condition in which microvesicles isolated from 1. mu.l or more body fluid are treated with 0.027AU or more protease; the 5 Xprotease concentration relates to an enzymatic condition in which microvesicles isolated from 1. mu.l or more of body fluid are treated with 0.135AU or more of protease; the 10 Xprotease concentration relates to an enzymatic condition in which microvesicles isolated from 1. mu.l or more of body fluid are treated with 0.27AU or more of protease; the 25 Xprotease concentration relates to an enzymatic condition in which microvesicles isolated from 1. mu.l or more of body fluid are treated with 0.675AU or more of protease; the 50 Xprotease concentration relates to an enzymatic condition in which microvesicles isolated from 1. mu.l or more of body fluid are treated with 1.35AU or more of protease. Preferably, the rnase inhibitor is a protease.

In yet another aspect, the invention is a novel kit for obtaining nucleic acids from microvesicles, comprising in one or more containers: (a) a nucleic acid extraction promoter; (b) dnase, rnase, or both; and (c) a lysis buffer. The novel kit may further comprise instructions for using the kit. In the novel kit of the present invention, the nucleic acid extraction promoter may include: in admixture or separately, (a) an rnase inhibitor, (b) a protease, (c) a reducing agent, (d) a decoy substrate, (e) a soluble receptor, (f) a small interfering RNA, (g) an RNA binding molecule, (h) an rnase-denaturing material, or (i) a combination of any of the foregoing.

In yet another aspect, the invention is a novel method of analyzing RNA from microvesicles, comprising the steps of: (a) obtaining a microbubble sample; (b) treating the sample with a DNase enzyme to remove all or substantially all of any DNA located outside or on the surface of the microvesicles in the sample; (c) extracting RNA from the sample; and (d) analyzing the extracted RNA. The novel method may be performed on a biological sample, including especially any body fluid, preferably urine, serum or plasma, preferably from a mammal, especially a human.

In a further aspect, the invention is novel for diagnosing, monitoring, or treating a subjectA method, comprising the steps of: (a) isolating a microvesicle fraction from a urine sample of a subject; (b) monitoring the microvesicle fraction for the presence of a biomarker; wherein the biomarker is selected from: (i) nucleic acids, (ii) nucleic acid expression levels, (iii) nucleic acid variants, and (iv) any combination of any of the foregoing; and wherein the biomarker is associated with the presence or absence of a disease or other medical condition, or the feasibility of a treatment option. In some aspects, the biomarker is an mRNA transcript; for example, the mRNA transcript may be selected from: NPHS2(podocin (an oligomer specifically expressed on the membrane of the foot process at the membrane of the foot cell fissure), LGALS1 (galectin-1), HSPG2 (heparin sulfate proteoglycan), CUBN (endocytosis receptor), LRP2 (megaprotein), AQP1 (aquaporin 1), CA4 (carbonic anhydrase 4), CLCN5 (chloride channel protein 5), BDKRB1 (bradykinin B1 receptor), CALCR (calcitonin receptor), SCNN1D (amiloride sensitive sodium channel subunit), SLC12A3 (thiazide sensitive sodium chloride cotransporter), AQP2 (aquaporin 2), ATP6V1B1 (V-atpase B1 subunit), SLC12a1 (kidney specific Na-K-C1 symporter via riboampednard-PCR); more preferably, the mRNA transcript is AQP2 (aquaporin 2) or ATP6V1B1 (V-ATPase B1 subunit). In a further aspect of the novel method, the biomarkers and diseases or other medical conditions are selected from: (a) NPHS2(podocin) and glomerular diseases such as hormone-resistant nephrotic syndrome; (b) such as Imerslund-CUBN (swallowing receptor) and proteinuria in syndrome (vitamin B12 selective malabsorption syndrome); and (c) AQP2 (aquaporin 2) and diabetes insipidus.

In yet another aspect, the invention is a polypeptide comprising a sequence selected from the group consisting of seq id nos: 1-29, an isolated polynucleotide molecule comprising a first nucleotide sequence having at least 90% identity to a second nucleotide sequence; comprises a sequence selected from SEQ ID NOS: 1-29; or comprises a polypeptide having a sequence identical to SEQ ID NOS: 1-29, and an isolated polynucleotide of a sequence of at least 13 nucleotides which is identical in any of the 13-nucleotide sequences. In particular, the aforementioned polynucleotide molecule may be a deoxyribonucleotide or a ribonucleotide. In other aspects, the invention is a vector comprising any of the aforementioned isolated nucleic acid molecules. In still other aspects, the invention is a host cell comprising any of the aforementioned vectors or any of the aforementioned isolated nucleic acids.

In a further aspect, the invention is a novel method of assessing the quality of a nucleic acid extract from a biological sample, the method comprising: (a) providing a biological sample; (b) obtaining a nucleic acid extract from the biological sample; (c) the extract was measured to contain a peptide having a sequence selected from the group consisting of SEQ NOS: 1-29, or a fragment thereof; and (d) comparing the amount of the polynucleotide molecule to a standard to assess the quality of the nucleic acid extract. The novel method can be performed on any biological sample, e.g. a body fluid, in particular urine, serum or plasma, preferably from a mammal such as a human. This novel method can be used in combination with any of the aforementioned novel nucleic acid extracts or novel extraction methods. In particular, the standard for evaluating the quality of nucleic acid extracts can be obtained by measuring the nucleic acid extracts from more than 5 biological samples containing nucleic acid sequences having sequences selected from seq id nos: 1-29, or a fragment thereof.

Drawings

In FIG. 1, the pictures a to f are electron microscope pictures of the urine polypole. The polypodies (MVBs) can be identified in various regions of the nephron and collecting duct (see arrows). Podo-podocyte, PT-proximal tubule, TDL-fine descending branch (thindecending limb), TAL-coarse ascending branch (thinckaindinglimb), CD-PC-ductus totalis main cell, CD-IC-ductus totalis leap cell. Scale bar 200nm for a, c, d, e, f; and 500nm for b.

Figure 2 is an electron microscope picture of isolated urine microbubbles. Human urinary microvesicles were isolated via differential ultracentrifugation and imaged via TEM using phosphotungstic acid as a staining agent. The scale bar is 200 nm.

FIG. 3 is a graph depicting an RNA profile generated using a 100kDaMWCO filter method. The graph was generated using an agilent bioanalyzer.

FIG. 4 is a graph depicting an RNA profile generated using the ultracentrifugation method. The graph was generated using an agilent bioanalyzer.

FIG. 5 is two graphs depicting RNA profiles generated using a three-step pretreatment procedure (A) of x300g spin, x17,000g spin, and 0.8 μm filtration, or a one-step pretreatment procedure with only 0.8 μm filtration (B), in each case followed by ultracentrifugation. The graph was generated using an agilent bioanalyzer.

FIG. 6 is two graphs depicting RNA profiles generated using a three-step pretreatment procedure (A) of x300g spin, x17,000g spin, and 0.8 μm filtration, or a one-step pretreatment procedure with only 0.8 μm filtration (B), in each case with filtration concentration immediately following treatment. The graph was generated using an agilent bioanalyzer.

FIG. 7 is a flow chart depicting a novel method for extracting nucleic acid from urine using an extraction-facilitating procedure.

FIG. 8 is two graphs depicting RNA profiles generated using the method using 5X and 10X concentrated protease. Microbubbles were isolated from a 20ml urine sample by a filter concentrator. Table A shows the map obtained using 5X protease. Table B shows the map obtained using 10 Xprotease. The 1X protease concentration relates to an enzymatic condition in which microvesicles isolated from 1 μ l or more of body fluid are treated with 0.027AU or more protease. The 5 Xprotease concentration relates to such enzymatic conditions as to those in which microvesicles isolated from 1. mu.l or more of body fluid are treated with 0.135AU or more of protease. 1mAU is the protease activity that releases folin positive amino acids and peptides equivalent to 1. mu. mol tyrosine per minute.

FIG. 9 is two graphs depicting RNA profiles generated using the method using 25X and 50X concentrated protease. Microbubbles were isolated from a 40ml urine sample by a filter concentrator. Table A shows the map obtained using 25X protease. Table B shows the map obtained using 50X protease. 1X protease refers to 0.027 AU. 1mAU is the protease activity that releases folin positive amino acids and peptides equivalent to 1. mu. mol tyrosine per minute.

Fig. 10 is a graph depicting the RNA profile of melanoma serum, sample 1. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

Fig. 11 is a graph depicting the RNA profile of melanoma serum, sample 2. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

Fig. 12 is a graph depicting the RNA profile of melanoma serum, sample 3. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

Figure 13 is a graph depicting the RNA profile of melanoma serum, sample 4. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

Fig. 14 is a graph depicting the RNA profile of melanoma serum, sample 5. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of rnase inhibitor was 3.2 units/. mu.l microbubble suspension buffer.

Fig. 15 is a graph depicting the RNA profile of melanoma serum, sample 6. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of rnase inhibitor was 3.2 units/. mu.l microbubble suspension buffer.

FIG. 16 is a graph depicting the RNA profile of normal serum, sample 7. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

FIG. 17 is a graph depicting the RNA profile of normal serum, sample 8. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

FIG. 18 is a graph depicting the RNA profile of normal serum, sample 9. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

FIG. 19 is a graph depicting the RNA profile of normal serum, sample 10. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of RNase inhibitor was 1.6 units/. mu.l microbubble suspension buffer.

FIG. 20 is a graph depicting the RNA profile of normal serum, sample 11. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of rnase inhibitor was 3.2 units/. mu.l microbubble suspension buffer.

FIG. 21 is a graph depicting the RNA profile of normal serum, sample 12. RNA was extracted from 1ml of serum by a method using RNase inhibitor, Superase-In (Ambion). The final concentration of rnase inhibitor was 3.2 units/. mu.l microbubble suspension buffer.

FIG. 22 is a flow chart depicting a novel method for extracting nucleic acids from a biological sample using an extraction-facilitating procedure.

FIG. 23 is two graphs depicting RNA profiles generated using the method with and without DNase treatment. Microvesicle particles isolated from urine samples are digested by dnase prior to lysis and nucleic acid extraction, removing DNA not located within the microvesicles. A-map generated using RNeasy Micro kit without DNase digestion. B-profile generated by DNase digestion using RNeasy Micro kit. C-map generated using the MirVana kit without DNase digestion. D-map generated by DNase digestion using the MirVana kit. Note the changes in small RNA peak height and area indicated by the arrows. D revealed that some DNA was carried into the sample after phenol/chloroform based extraction.

FIG. 24 is two graphs depicting RNA profiles generated using the method with and without DNase treatment. Microvesicle particles isolated from urine samples are digested by dnase prior to lysis and nucleic acid extraction, removing DNA not located within the microvesicles. A-is a pattern generated without DNase digestion. B-map generated by DNase digestion. C-a pseudo-gel exhibiting an "apoptotic body" like ladder that can co-segregate with serum-derived microvesicles.

FIG. 25 is two graphs depicting RNA profiles generated using the method with and without RNase treatment. Microvesicle particles isolated from urine samples are digested by rnase prior to lysis and nucleic acid extraction to remove RNA not located within the microvesicles. A-a pattern generated without RNase digestion. B-profile generated by RNase digestion.

FIG. 26 is two graphs depicting RNA profiles generated from urine microvesicles and rat kidney tissue. A-map from rat kidney tissue. B-profile from urinary microvesicles.

FIG. 27 is two graphs of RNA profiles generated from urinary microvesicles and rat kidney tissue using a method that can enrich for small RNA extracts. A-map from rat kidney tissue. B-profile from urinary microvesicles.

FIG. 28 is two graphs depicting RNA profiles generated from whole urine with or without DNase treatment from which microvesicles have been removed and have not been captured by isolation techniques. A-nucleic acids were isolated from whole urine without DNase treatment. B-nucleic acids isolated from whole urine by DNase treatment.

Figure 29 is two graphs depicting RNA profiles generated from urinary microvesicles. A-nucleic acids isolated from urine microvesicles without DNase treatment. B-nucleic acids isolated from urine microvesicles by DNase treatment.

FIG. 30 is two graphs depicting RNA profiles generated from nucleic acids extracted from particles formed during 300g rotation. A-nucleic acids isolated from 300g of spun particles without DNase treatment. B-nucleic acids isolated from 300g of spun particles by DNase treatment.

FIG. 31 is two graphs depicting RNA profiles generated from nucleic acids extracted from particles formed during 17,000g rotation. A-nucleic acid profile generated from 17,000g of spun particles without DNase treatment. B-nucleic acid profile generated from 17,000g of spun particles by DNase treatment.

Figure 32 is two graphs depicting RNA profiles generated by microvesicles that have been externally subjected to rnase and dnase digestion, with or without intra-microvesicle rnase digestion, prior to microvesicle lysis. A-nucleic acid profile generated without enzymatic digestion of RNA in the microvesicles. B-nucleic acid profile generated by RNase digestion in microvesicles.

Figure 33 is two graphs depicting RNA profiles generated by microvesicles that have been externally subjected to rnase and dnase digestion, with or without dnase digestion within the microvesicles, prior to microvesicle lysis. A-nucleic acid profile without digestion with DNase in the microvesicles. B-nucleic acid profile generated by DNase digestion within the microvesicles. On graph B, the peak immediately after 20 seconds is reduced compared to the matching peak in graph a. This reduction indicates that a small amount of DNase digestible material is present within the exosome.

FIG. 34A) is a "pseudo-gel" profile generated by BioAnalyzer by RT-PCR analysis of urinary microvesicles, in which mRNA transcripts of RiboAmp amplified β -actin and GAPDH could be unambiguously identified; B) the nephron and collecting catheter are shown highlighting its six functionally distinct regions. 1. Glomeruli; 2. a proximal tubule; 3. fine descending branches; 4. thick ascending branches of medulla; 5.a distal convoluted tubule; 6. a collection conduit.

FIG. 35 shows a pseudogel profile generated by a BioAnalyzer, detected by RT-PCR analysis of RiboAmpedmRNA from urinary microvesicles, in which mRNA transcripts from regions 1 and 2 of the nephron and ductus effervesces encoding specific genes could be identified, in particular: 1. glomeruli: NPHS2-podocin (oligomer specifically expressed on the membrane of the podocyte apopore membrane), LGALS 1-galectin-1, HSPG 2-heparan sulfate proteoglycan. 2. Proximal tubule: CUBN-gulp receptor, LRP 2-megaprotein, AQP 1-aquaporin 1, CA 4-carbonic anhydrase 4, CLCN 5-chloride channel protein 5.

FIG. 36 shows a pseudogel profile generated by BioAnalyzer, detected by RT-PCR analysis of RiboAmpedmRNA from urinary microvesicles, in which mRNA transcripts from regions 3-6 of the nephron and ductus effervesces encoding specific genes could be identified, in particular: 3. fine descending branches: BDKRB 1-bradykinin B1 receptor. 4. Thick ascending branches of medulla: CALCR-calcitonin receptor, SCNN 1D-amiloride sensitive sodium channel subunit. 5. Distal convoluted tubule: SLC12A 3-thiazide-sensitive sodium chloride cotransporter. 6. A collecting conduit: AQP 2-aquaporin 2, ATP6V1B1-vATPaseB1 subunit, SLC12A 1-kidney specific Na-K-C1 symporter.

FIG. 37A) are two BioAnalyzer pseudo-gel profiles depicting expression of V-ATPaseB1KO (B1-/-) and wild type (B1+/+) V-ATPaseB1 subunit and AQP2mRNA in mice detected by RT-PCR analysis; B) are two graphs depicting expression of the V-ATPaseB2 subunit in V-ATPaseB1KO (B1-/-) and wild-type (B1+/+) mouse urine microvesicles and kidney cells, detected by real-time PCR analysis. "NS" -has no statistical significance.

FIG. 38 is three graphs depicting RNA profiles produced by urinary microvesicles. The microvesicles were not washed or treated with any extraction promoters before disrupting the microvesicle membrane for nucleic acid extraction. Three samples were used in this set of extractions. The maps are shown in fig. A, B and C, respectively.

FIG. 39 is three graphs depicting RNA profiles produced by urinary microvesicles. Prior to disrupting the microvesicle membrane for nucleic acid extraction, the urinary microvesicles were not washed but treated with RNase inhibitor, RNase-in (Promega). Three samples were used in this set of extractions. The maps are shown in fig. A, B and C, respectively.

FIG. 40 is three graphs depicting RNA profiles produced by urinary microvesicles. Urine microvesicles were washed but not treated with any rnase inhibitor before the microvesicle membranes were disrupted for nucleic acid extraction. Three samples were used in this set of extractions. The maps are shown in fig. A, B and C, respectively.

FIG. 41 is three graphs depicting RNA profiles produced by urinary microvesicles. Urine microvesicles are washed and treated with rnase inhibitors before the microvesicle membranes are disrupted for nucleic acid extraction. Three samples were used in this set of extractions. The maps are shown in fig. A, B and C, respectively.

Fig. 42 is a list of chromosomal regions where there were more than 500 transcript results ("spikes") in deep sequencing analysis of RNA extracted from urine microvesicles. The numbers indicate the start and end points of each chromosomal region. For example, "chr 1. -1.91625366.91625741" refers to the region on human chromosome 1 between nucleotides 91625366 and 91625741. The corresponding SEQ ID NOs are also indicated.

FIG. 43 is a list of primers used in PCR reactions to amplify sequences in the 10 chromosomal regions indicated. For example, "chr 1. -1.91625366.91625741" refers to the region on human chromosome 1 between nucleotides 91625366 and 91625741. The primer pairs used to amplify this region were "tccagctcacgttccctatt 1L and ccaggtggggagtttgact 1R". The primers extend from left to right from 5 'to 3'.

FIG. 44 is two BioAnalyzer pseudo-gel profiles depicting the results of PCR amplification of 10 peak-rich chromosomal regions. The lane numbers at the top of each figure correspond to the numbers of the chromosomal regions shown in figure 43. In A, nucleic acid extracts from urine microvesicles were used as PCR templates. In B, nucleic acid extracts from kidney tissue were used as PCR templates.

FIGS. 45-73 are graphs depicting spikes in 29 chromosome regions. This region is indicated at the top of each graph. For example, the graph in fig. 46 indicates the region "chr 1" -1.91625366.91625741 ", which is the region on human chromosome 1 between nucleotides 91625366 and 91625741.

Detailed Description

Microvesicles are shed by eukaryotic cells or bud from the plasma membrane to the outside of the cell. These membrane vesicles are non-uniform in diameter and have a size of about 10nm to about 5000 nm. Small microvesicles (about 10 to 1000nm in diameter, more often about 10 to 200nm) released by intracellular multivesicular exocytosis are known in the art as "exosomes". The compositions, methods and uses described herein are equally applicable to microbubbles of all sizes, preferably 10 to 800nm, more preferably 10 to 200 nm.

In some literature, the term "exosomes" is also referred to as protein complexes containing exoribonucleases involved in mRNA degradation and nucleolar small rna (snorna), intranuclear small rna (snrna), and ribosomal rna (rrna) processing (Liu et al, 2006; vanDijk et al, 2007). Such protein complexes do not have a membrane, and are not those terms "microvesicles" or "exosomes" as used herein.

The present invention is based in part on this discovery: adverse factors can prevent efficient extraction of nucleic acids from biological samples, and new and unexpected agents and procedures can be used to mitigate or eliminate the adverse factors, thereby significantly improving the quality of the extracted nucleic acids. Accordingly, one aspect of the present invention is a novel method for extracting high quality nucleic acids from a biological sample. The high quality extracts obtained by the novel process described herein are characterized by high yield and high integrity, making the extracted nucleic acids useful for a variety of applications, preferably high quality nucleic acid extracts.

In general terms, the new method comprises, for example, the following steps: obtaining a biological sample, mitigating or removing adverse factors that prevent efficient extraction of nucleic acids from the biological sample, and extracting nucleic acids from the biological sample, optionally followed by nucleic acid analysis.

Applicable biological samples include, for example, a cell, a group of cells, a cell fragment, a cell product including, for example, microvesicles, a cell culture, a body tissue from a subject, or a body fluid. The body fluid may be a fluid isolated from any part of the subject's body, preferably the peripheral region, including, but not limited to, blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluids of the respiratory, intestinal and genitourinary tracts, tears, saliva, breast milk, lymphatic system fluid, semen, cerebrospinal fluid, intra-organ systemic fluid, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof.

The biological sample may sometimes be from a subject. The term "subject" is intended to include all animals that show or are expected to have microvesicles. In particular embodiments, the subject is a mammal, human or non-human primate, dog, cat, horse, cow, other farm animal, or rodent (e.g., mouse, rat, guinea pig, etc.). The terms "subject" and "individual" are used interchangeably herein.

The biological sample may optionally be processed to obtain a biological sample derivative before, after, or simultaneously with the step of mitigating or removing the adverse effect. The biological sample derivative may be a cell, a cell fragment, a membrane vesicle, or a microbubble.

Biological samples are sometimes pre-processed prior to obtaining derivatives of the biological sample, such as microbubbles. In some cases, this preprocessing step is preferred. For example, a urine sample may be pre-processed to obtain urine microbubbles. This pre-processing can be accomplished by techniques known in the art such as low speed centrifugation and pre-filtration. For example, a urine sample may be subjected to a first centrifugation step of 300g to remove large particles from the sample. The urine sample may be subjected to a second centrifugation step at 17,000g to remove smaller particles from the sample. After the second centrifugation step, the urine sample may be further subjected to a pre-filtration step, e.g., a 0.8um pre-filtration step. Alternatively, the urine sample may be pre-processed by a pre-filtration step without being subjected to one or more of the centrifugation steps.

Membrane vesicles, such as microbubbles, can be obtained from a biological sample. In some cases, such separation may be performed in some cases without pre-processing the biological sample. In other cases, such separation may be performed after pre-processing the biological sample. This separation step may be advantageous for high quality nucleic acid extracts from biological samples. For example, the separation may yield advantages such as: 1) there is the possibility of selectively analysing disease or tumour specific nucleic acids which can be obtained by separating disease or tumour specific microvesicles from other microvesicles in a fluid sample; 2) the obtained nucleic acids have a significantly higher yield and higher integrity compared to the yield/integrity obtained by extracting the nucleic acids directly from the fluid sample; 3) scalability, e.g., to detect nucleic acids expressed at low levels, may increase sensitivity by granulating more microbubbles from a larger volume of serum; 4) nucleic acids are relatively pure because proteins and lipids, debris of dead cells, and other potential contaminants and PCR inhibitors are removed from the microvesicle particles prior to the nucleic acid extraction step; and 5) more options in nucleic acid extraction methods, because the microbubble particles are much smaller than the starting serum volume, making it possible to extract nucleic acids from these microbubble particles using a small volume column filter.

Methods for isolating microvesicles from a biological sample are known in the art. For example, differential centrifugation methods are described in the paper of Raposo et al (Raposo et al, 1996), Skog et al (Skog et al, 2008), and Nilsson et al (Nilsson et al, 2009). Methods of anion exchange and/or gel permeation chromatography are described in U.S. patent nos. 6,899,863 and 6,812,023. Methods for sucrose density gradient or organelle electrophoresis are described in U.S. patent No. 7,198,923. Methods of Magnetic Activated Cell Sorting (MACS) are described in the articles by Taylor and Gercel-Taylor (Taylor and Gercel-Taylor, 2008). The nano-membrane ultrafiltration concentration method is described in the article by Cheruvanky et al (Cheruvanky et al, 2007). Further, microvesicles can be identified and isolated from a subject's body fluid by newly developed microchip technology that efficiently and selectively isolates tumor-derived microvesicles using a unique microfluidic platform (Chen et al). Each of the foregoing references is incorporated herein by reference for the purpose of teaching these methods.

In one embodiment of the methods described herein, in microvesicles isolated from a bodily fluid, those originating from a particular cell type, e.g., lung, pancreas, stomach, small intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, fetal cells, are enriched. Because the microvesicles typically carry surface molecules such as antigens from their donor cells, the surface molecules can be used to identify, isolate and/or enrich for microvesicles from a particular donor cell type (Al-Nedawi et Al, 2008; Taylor and Gercel-Taylor, 2008). In this way, microvesicles derived from different cell populations can be analyzed for nucleic acid content. For example, tumor (malignant and non-malignant) microvesicles carry surface antigens associated with tumors and can be detected, isolated and/or enriched by surface antigens associated with these specific tumors. In one example, the surface antigen is an epithelial cell adhesion molecule (EpCAM) that is specific for microvesicles from lung, colorectal, breast, prostate, head and neck, and liver origin, but not for microvesicles from blood cell origin (Balzar et al, 1999; Went et al, 2004). In another example, the surface antigen is CD24 and CD24 is a glycoprotein specific for urinary microvesicles (Keller et al, 2007). In yet another example, the surface antigen is selected from the group of molecules such as CD70, carcinoembryonic antigen (CEA), EGFR, EGFRvIII, and other variants, Fas ligand, TRAIL, transferrin receptor, p38.5, p97, and HSP 72. In addition, tumor-specific microvesicles are characterized by the absence of surface markers, such as CD80 and CD 86.

Isolation of microvesicles of a particular cell type can be accomplished, for example, by using antibodies, aptamers, aptamer analogs or molecularly imprinted polymers specific for the desired surface antigen. In one embodiment, the surface antigen is specific for a type of cancer. In another embodiment, the surface antigen is specific for a cell type that is not necessarily cancerous. An example of a cell surface antigen based microbubble isolation method is provided in U.S. patent No. 7,198,923. Aptamers and analogs thereof specifically bind to surface molecules and can be used as isolation tools to recover cell type-specific microvesicles, as described in U.S. Pat. nos. 5,840,867 and 5,582,981, WO/2003/050290, and publications of Johnson et al (Johnson et al, 2008). Molecularly imprinted polymers also specifically recognize surface molecules, as described in U.S. patent nos. 6,525,154, 7,332,553, and 7,384,589 and publications by Bossi et al (Bossi et al, 2007), and are tools for recovering and analyzing cell-type specific microvesicles. Each of the foregoing references is incorporated herein by reference for the purpose of teaching these methods.

In the case where the desired biological derivative is a membrane vesicle such as a microbubble, a step of removing nucleic acids not located within the microbubble is sometimes performed. Methods for removing nucleic acids are well known in the art. For example, to remove such nucleic acids from a sample, an enzymatic digestion step may be performed. Such enzymes can be of the ribonuclease type which catalyzes ribonuclease digestion, or of the deoxyribonuclease type which catalyzes deoxyribonuclease digestion.

In one aspect of the invention, the novel nucleic acid extraction method includes a step of removing or mitigating adverse factors that prevent high quality extraction of nucleic acids in a biological sample. Such adverse factors are diverse, as different biological samples may contain various kinds of such adverse factors. In some biological samples, factors such as excess extra-microvesicle DNA may affect the quality of nucleic acid extracts from such samples, contaminating DNA extracted from the microvesicles. In other samples, factors such as excess endogenous rnase may affect the quality of nucleic acid extracts from such samples. A number of agents and methods may be used to remove these disadvantages. These methods and agents are collectively referred to as "extraction promotion operation".

In some cases, the extraction-facilitating operation may include adding a nucleic acid extraction facilitating agent to the biological sample or to the derivative. To remove adverse factors such as endogenous rnases, such extraction promoters as defined herein may include, but are not limited to, commercially available rnase inhibitors such as superfase-In (ambion inc.), RNaseIN (PromegaCorp.) or other agents that function In a similar manner, proteases, reducing agents, decoy substrates such as synthetic RNA, soluble receptors that bind rnases, small interfering RNA (sirna), RNA binding molecules such as anti-RNA antibodies or chaperones, rnase denaturing substances such as hypertonic solutions, detergents, or combinations thereof. These promoters may function in various ways, such as, but not limited to, by inhibiting rnase activity (e.g., rnase inhibitors), by degradation that is prevalent in proteins (e.g., proteases), or by chaperones that bind and protect RNA (e.g., RNA-binding proteins). In all cases, such an extraction promoter can remove or mitigate some or all of the disadvantages of a biological sample that would otherwise prevent or interfere with high quality extraction of nucleic acids from the biological sample.

In other cases, the extraction enhancement operation may include performing one or more processing steps. Such processing includes washing the nucleic acid-containing components of the sample, such as microbubbles, sufficiently or substantially completely; size sorting (sizing) the rnase of the biological sample; proteins in a biological sample are denatured by a variety of techniques including, but not limited to, generating specific pH conditions, temperature conditions (e.g., reduced or maintained at a lower temperature), freeze/thaw cycles, and combinations thereof.

One striking manifestation of the use of the extraction-facilitated procedures described herein is the ability to detect the presence or absence of significant amounts of ribosomal rna (rrna) in nucleic acid extracts of microvesicles. There is currently no study available to demonstrate that 18SrRNA and 28SrRNA are detected in microvesicle nucleic acid extracts. In contrast, prior studies have suggested that there is no or only very little rRNA present in nucleic acid extracts of microvesicles (Skog et al, 2008; Taylor and Gercel-Taylor, 2008; Valadi et al, 2007).

In another aspect of the invention, the performance of the extraction-facilitating operation will improve the quality of the extracted RNA in terms of RNA integrity (RNA quality integrity index, RIN). RNA Integrity (RIN), designed by Agilent technologies (http:// www.chem.agilent.com/en-us/products/instruments/lab-on-a-chip/pages/gp14975.aspx, 15.7.2010 visit), is the product of a software tool designed to evaluate the integrity of total RNA samples. The software automatically assigns integrity to total RNA samples of eukaryotes. With this tool, sample integrity is not determined by the ratio of 18S and 28S ribosomal bands, but by the entire electrophoretic trace (trace) of the RNA sample. This includes the presence or absence of degradation products. The assigned RIN is independent of sample concentration, instrumentation, and analysts and can be used as a standard for RNA integrity.

In yet another aspect of the invention, the performance of the extraction-facilitating operation will increase the amount or yield of extracted nucleic acids. For example, nucleic acid yields of greater than or equal to 50pg/ml can be obtained from 20ml of a low protein biological sample, such as urine, using extraction-facilitating procedures as described herein. Alternatively, a nucleic acid yield of greater than or equal to 50pg/ml can be obtained from 1ml of a high protein biological sample such as serum or plasma.

The novel high quality nucleic acid extracts obtained by the methods described herein may exhibit a combination of 18S rRNA and 28S rRNA in a ratio of about 1: 1 to about 1: 2, preferably about 1: 2, as detected; an RNA integrity of greater than or equal to 5 for a low protein biological sample, or greater than or equal to 3 for a high protein biological sample; and a nucleic acid yield greater than or equal to 50pg/ml from 20ml of the low protein biological sample or 1ml of the high protein biological sample.

High quality RNA extracts are highly desirable because RNA degradation can severely affect the extracted RNA, such as in gene expression and mRNA analysis, as well as downstream evaluation in non-coding RNAs such as small RNAs and microRNA analysis. The novel methods described herein enable one to extract high quality nucleic acids from biological samples, such as microvesicles, so that gene expression and mutation levels within exosomes can be accurately analyzed. In one embodiment, for example, when increasing the concentration of protease (5X, 10X) used as an extraction promoter, the amount and integrity of RNA isolated from urine microvesicles increases significantly.

In another aspect of the invention, methods are provided for extracting high quality small RNA from a biological sample, such as urine. Small RNAs, such as mirnas, are particularly susceptible to degradation and loss during nucleic acid extraction. In the novel methods disclosed herein, the use of high concentrations of protease removes or mitigates the disadvantages that prevent high quality extraction of small RNAs. In one example, a method of extracting nucleic acids, particularly small RNAs, using 25X and 50X proteases as extraction promoters, significantly increased amounts of small RNAs can be obtained. As used herein, expression such as 5X, 10X, 25X, and 50X refers to levels of protease activity that are 5 times, 10 times, etc. greater than the levels of protease activity currently used or recommended in commercially available nucleic acid extraction kits, such as the qiaampminelutevirussspin kit.

When the adverse factors affecting extraction have been removed or mitigated, the nucleic acid molecules can be isolated from the biological sample using a number of procedures well known in the art. One skilled in the art will be able to select a particular isolation procedure appropriate for a particular nucleic acid sample. Examples of extraction methods are provided in the examples section herein. In some cases, nucleic acids can also be analyzed without extraction from the microvesicles using some techniques.

In one embodiment, the extracted nucleic acids, including DNA and/or RNA, can be analyzed directly without an amplification step. Direct analysis can be performed using different methods, including, but not limited to, nanochain (nanostring) techniques. Nanostring technology enables one to identify and quantify individual target molecules in a biological sample by attaching color-coded fluorescent indicators to each target molecule. This approach is similar to the concept of measuring inventory by scanning bar codes. The indicator may be made of hundreds or even thousands of different codes that allow for highly multiplexed analysis. This technique is described in a publication by Geiss et al (Geiss et al, 2008) and is incorporated herein by reference for teaching purposes.

In another embodiment, it may be advantageous or otherwise desirable to amplify the nucleic acid of the microvesicles prior to analyzing it. Nucleic acid amplification methods are common and well known in the art, many examples of which are described herein. If desired, the amplification may be performed such that it is quantitative. Quantitative amplification will allow the relative amounts of the various nucleic acids to be quantitatively determined to produce a profile as described below.

In one example, the extracted nucleic acid is RNA. Then, prior to further amplification of the RNA, it is preferably reverse transcribed into complementary DNA (cDNA). Such reverse transcription may be performed alone or in combination with an amplification step. One example of a method that combines reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which can be further modified to be quantitative, such as the quantitative RT-PCR described in U.S. Pat. No. 5,639,606, which is incorporated herein by reference for teaching purposes.

Nucleic acid amplification methods include, but are not limited to, Polymerase Chain Reaction (PCR) (U.S. patent No. 5,219,727) and variants thereof such as in situ polymerase chain reaction (U.S. patent No. 5,538,871), quantitative polymerase chain reaction (U.S. patent No. 5,219,727), nested polymerase chain reaction (U.S. patent No. 5,556,773), autonomous sequence replication and variants thereof (Guatelli et al, 1990), transcription amplification systems and variants thereof (Kwoh et al, 1989), Qb replicase and variants thereof (Miele et al, 1983), cold PCR (Li et al, 2008), or any other nucleic acid propagation method followed by detection of the amplified molecules using techniques well known to those skilled in the art. Those detection schemes designed for the detection of nucleic acid molecules are particularly useful if such molecules are present in very small numbers. The foregoing references are incorporated herein by reference for the purpose of teaching these methods.

The analysis of the nucleic acids present in the microvesicles is quantitative and/or qualitative. For quantitative analysis, the amount (expression level) of a particular nucleic acid of interest within a microvesicle, whether relative or absolute, can be measured using methods known in the art (described below). For qualitative analysis, the particular nucleic acid species of interest within the microvesicles, whether wild-type or variants thereof, can be identified using methods known in the art.

The invention disclosed herein also includes nucleic acid extracts from microvesicles as a novel composition of matter, wherein 18s rrna and 28s rrna can be detected in the extract. Such nucleic acid extraction can be accomplished using the novel nucleic acid extraction methods disclosed herein. High quality nucleic acid extraction of microvesicles in biological samples is desirable in many cases. In some cases, tissue samples are not readily available (eacidacesible). For example, brain tumor samples are not usually obtained without brain surgery. In contrast, microvesicles samples from sera of patients with brain tumors are readily available. In order to analyze nucleic acids in brain tumor cells, it is easier to analyze nucleic acids in serum microvesicles secreted from brain tumor cells. Therefore, in the case of replacing the nucleic acid from the tissue cells with the nucleic acid in the microvesicles secreted from the tissue cells, it is desirable to obtain a high quality nucleic acid containing detectable quality control substances (qualitycontrols) such as 18SrRNA and 28SrRNA like those directly obtained from the tissue cells. In other cases, high quality small RNAs are desirable. The nucleic acid extracts disclosed herein comprise such high quality small RNAs, as well as 18SrRNA and 28 SrRNA. Such high quality small RNAs are important for accurately assessing the expression levels of nucleic acids for various purposes, e.g., specific mirnas.

The invention disclosed herein further includes novel high quality profiles of nucleic acids from microvesicles in a biological sample. Such profiles are generated by analyzing a nucleic acid extract comprising 18SrRNA and 28 SrRNA. Such a profile can be obtained using the novel methods disclosed herein. High quality nucleic acid profiles are highly desirable for many applications, such as applications as markers of choice for medical conditions or therapies. Such a profile is expected to be consistent between different samples. Such consistency is hardly obtainable in the absence of high quality nucleic acid extracts. In one embodiment of the invention, a nucleic acid profile may be obtained by analyzing the nucleic acids in microvesicles secreted by cells of these origins. Such microvesicles can be isolated from readily available biological samples, such as urine, serum or plasma. Such nucleic acid maps may include small RNAs, messenger RNAs, micrornas, non-coding RNAs, or combinations thereof. In a further embodiment of the invention, such a nucleic acid profile may be combined with other biomarkers to obtain certain results more accurately.

The nucleic acid profile, for example, can be a collection of genetic aberrations that are used herein to refer to the amount of nucleic acid within the microvesicle, as well as nucleic acid variants. Specifically, genetic aberrations include, but are not limited to, overexpression of a gene (e.g., an oncogene) or set of genes, under-expression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or set of genes, generation of alternative splice variants of a gene or set of genes, gene Copy Number Variation (CNV) (e.g., DNA duplex) (Hahn, 1993), nucleic acid modifications (e.g., methylation, acetylation, and phosphorylation), Single Nucleotide Polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions, and duplications), and mutations (insertions, deletions, duplications, mismeanings, nonsense, synonyms, or any other nucleotide changes) of a gene or set of genes that in many cases ultimately affect the activity and function of the gene product, result in the formation of alternative transcriptional splice variants, and/or result in altered levels of gene expression.

Such genetic aberrations can be determined by a variety of techniques known to the skilled artisan. For example, nucleic acid expression levels, alternative splice variants, chromosomal rearrangements, and gene copy number can be determined by microarray technology (U.S. Pat. nos. 6,913,879, 7,364,848, 7,378,245, 6,893,837, and 6,004,755) and quantitative PCR. In particular, copy number changes can be detected using IlluminaInfiniumII whole genome genotyping analysis or Agilent HumangenomeCGHMicroarray (Steemers et al, 2006). Nucleic acid modifications can be analyzed by methods described in, for example, U.S. Pat. No. 7,186,512 and patent application WO/2003/023065. In particular, methylation profiles can be determined by IlluminaDNAlhylationOMA 003cancer Panel. SNPs and mutations can be detected by hybridization using allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatched heteroduplexes (Cotton et al, 1998), ribonuclease cleavage of mismatched bases (Myers et al, 1985), mass spectrometry (U.S. Pat. Nos. 6,994,960, 7,074,563 and 7,198,893), nucleic acid sequencing, single-strand conformation polymorphism (SSCP) (Orita et al, 1989), Denaturing Gradient Gel Electrophoresis (DGGE) (Fischer and Lerman, 1979 a; Fischer and Lerman, 1979b), Temperature Gradient Gel Electrophoresis (TGGE) (Fischer and Lerman; 1979 a; Fischer and Lerman, 1979b), Restriction Fragment Length Polymorphism (RFLP) (Kan and Dozy, 1978 a; Kan and Dozy, 1978b), Oligonucleotide Ligation Assays (OLA), allele-specific PCR (ASPCR) (U.S. Pat. No. 5,639,611), ligase chain reaction (Abraya) and variants thereof (Abraya et al, 1995; Nakazaen et al, 1988; Nakazan et al, 1994) flow cytometry heteroduplex analysis (WO/2006/113590) and combination/modification detection thereof. In particular, gene expression levels can be determined by Serial Analysis of Gene Expression (SAGE) technology (Velculescu et al, 1995). Generally, methods for analyzing genetic aberrations are reported in numerous publications, not limited to those cited herein, and are available to the skilled artisan. Appropriate methods of analysis depend on the specific target of the analysis, the patient's condition/medical history, and the particular cancer (or cancers) to be detected, monitored or treated, the disease or other medical condition. The foregoing references are incorporated herein for the purpose of teaching these methods.

It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

Embodiments of the presently disclosed subject matter are presented below. Other features, objects, and advantages of the disclosed subject matter will be apparent from the description and drawings, and from the embodiments and from the claims. The presently disclosed subject matter may be practiced or tested with methods, devices, and materials substantially similar or equivalent to those described herein. Example methods, devices, uses and materials are described below.

Microbubbles in urine

Example 1: renal cells contain polyvacuoles

To examine whether kidney cells shed microvesicles, we used Transmission Electron Microscopy (TEM) to determine whether kidney cells contain microvesicles that can produce microvesicles. Rat kidneys were fixed by intravascular perfusion with 2.0% glutaraldehyde in 7.4 sodium cacodylate buffer at 0.1MpH (Electron Microscopysciences, Pa.), and kidney sections were further fixed overnight at 4 ℃. The sample sections were washed with 0.1M sodium cacodylate buffer, fixed with 1.0% osmium tetroxide in cacodylate buffer for 1h at room temperature, washed again with buffer, and then washed with distilled water (dH)₂O) rinsed and the monolith (enbloc) was stained in 2.0% uranyl acetate in water for 1 hour at room temperature. The sample was rinsed with distilled water and dehydrated to 100% by gradient ethanol. Epon resin (TedPella, CA) infiltration of the sample was accomplished by soaking the sample overnight in a 1: 1 solution of Epon: ethanol. The next day the samples were placed in fresh Epon for several hours and buried overnight in Epon at 60 ℃. Thin sections were cut on Reichert UltracutEultmicotome, collected on a polyvinyl formal (formvar) coated mesh, and stained with uranyl acetate and lead citrate. The samples were examined under a JEOLJEM1011 transmission electron microscope at 80 kV. Images were collected using an AMT (advanced microscopical techniques, MA) digital imaging system. As shown in fig. 1, a Transmission Electron Microscope (TEM) image of MVB was seen in rat kidney tissue cells. Multivesicular bodies (MVBs) can be identified in various regions of the nephron and duct, including podocytes, proximal tubules, fine descending branches, rough ascending branches, duct main cells, and duct leap cells. This demonstrates that exosomes can indeed be released in various regions of the nephron and in the intercalated and primary cells of the collecting duct.

Example 2: presence of microbubbles in urine

To examine the microbubbles themselves, we examined human urine microbubbles by TEM. Human urine was obtained according to the IRB guidelines approved by Massachusetts general Hospital. The urine is then pre-processed by a process consisting of three steps: the urine was centrifuged at 300g for 10 minutes at 4 ℃, the supernatant was centrifuged at 17,000g for 20 minutes at 4 ℃, and the supernatant was filtered through a 0.8 μm filter (nitrocellulose membrane filter device, Nalgene, NY). Alternatively, the pre-processed urine may be filtered by one-step filtration, directly through a 0.8 μm filter, without performing any pre-centrifugation step. In either case, the filtrate was then ultracentrifuged at 118,000g for 70 minutes at 4 ℃, the supernatant removed, the microvesicle-containing particles washed in PBS, and the beadlets reconstituted at 118,000g for 70 minutes at 4 ℃.

Filtration concentration can also be used instead of centrifugation to separate microbubbles from the pre-processed sample. The filtration concentrator (100kDamWC CO) (Millipore, MA) was prepared according to the manufacturer's instructions. The pre-processed filtrate was added to a filtration concentrator and centrifuged at 4,000g for 4 minutes at RT. A15 ml PBS wash step was included.

In dH at a ratio of 1: 1 of 4%₂Paraformaldehyde in O immobilizes the microbubble particles. Ten (10) μ l drops were pipetted into formvar coated 200 mesh gold mesh and withdrawn after 1 minute. By dH₂O drops wash the samples 2 times. Aqueous 2.0% phosphotungstic acid (PTA) (10. mu.l) was applied for 10 seconds, extracted and dH used₂And flushing once. The samples were examined under a JEOLJEM1011 transmission electron microscope at 80 kV. Images were collected using an AMT (advanced Microcopy technologies, Mass.) digital imaging system. As shown in fig. 2, the particles are indeed enriched in microbubbles. The microbubbles sometimes cluster together or remain individual in the TEM image.

Improved method for extracting nucleic acid from biological sample

Example 3: the filter concentrator can replace ultracentrifugation for microbubble separation

We demonstrate here that similar to the ultracentrifugation method, the filter concentrator can also generate viable (viable) microvesicles for RNA extraction. We preprocessed the 75ml urine as described in detail in example 2 by centrifugation at 300g for 10 minutes at 4 ℃ and 17,000g for 20 minutes at 4 ℃ followed by filtration through a 0.8 μm filter. Microvesicles were then isolated by a 100kDaMWCO filtration concentrator (Millipore, MA) and by ultracentrifugation, both methods using RNase digestion and with and without DNAse digestion to remove nucleic acid contamination outside the microvesicles, respectively. As shown in FIGS. 3 and 4, the ultracentrifugation method and the filtration concentration method obtained similar RNA concentrates from 75ml urine samples, wherein the ultracentrifugation (FIG. 4) was 410. + -.28 pg/. mu.l and the filtration concentrator (FIG. 3) was 381. + -.47 pg/. mu.l (mean. + -. SD). There was no statistically significant difference between these two yields. These data demonstrate that the use of a filter concentrator is a reliable method for the aerosolization of urinary microbubbles for RNA analysis.

Example 4: the method of pre-treating the sample with only 0.8um pre-filtration step is sufficient for microbubble isolation

Further, we found that the low speed centrifugation step at 300g and 17,000g could be eliminated, since the method of pre-processing urine with only 0.8um pre-filtration step is as effective as the method comprising the low speed centrifugation step. As shown in fig. 5 and 6, the nucleic acid pattern (a) generated using the low-speed centrifugation together with the 0.8um prefiltering method was the same as the pattern (B) generated using the 0.8um prefiltering method alone.

Example 5: extraction of nucleic acids from urine microvesicles using a method comprising removal or mitigation of adverse factors

We utilized an improved method of nucleic acid extraction from microvesicles. In this method, we removed the adverse factors for high quality nucleic acid extraction prior to disrupting the microbubble membrane. As shown in fig. 7, a pre-processed urine sample 100 is filtered by passing through a 0.8 μm filter membrane 110. The microbubbles 120 in the filtrate are then isolated by ultracentrifugation or concentration by filtration, the details being similar to those described in example 2. The isolated microvesicles are then subjected to rnase and/or dnase digestion to remove nucleic acids 130 not contained inside the microvesicles. Specifically, the microvesicles were resuspended in 1. mu.l/ml RNase A (DNase and protease free) (Fermentas, MD) in PBS and incubated at 37 ℃ for 1 hour. To perform the DNase I digestion, the pellet was resuspended in 500. mu.l PBS and DNase I (No RNase) (Qiagen, CA) diluted (according to the manufacturer's instructions) in RDD buffer and incubated for 10 minutes at room temperature. The sample was re-pelleted in PBS at 118,000g for 70 minutes. To perform rnase a and dnase I digestion of microvesicles isolated via the filter concentrator, incubation was performed in the filter concentrator using the same concentrations of rnase and dnase. Extraction facilitation 140 steps are then performed, e.g., three resuspension/washing steps with 15ml pbs alone or in combination with protease treatment. After microbubble isolation, nucleic acid 150 is extracted by nuclease digestion and protease treatment. RNA was extracted using RNeasyMicro kit (Qiagen, CA) according to the manufacturer's instructions. Briefly, exosomes were lysed using 350. mu.l of RLT buffer (with 10. mu.l β -mercaptoethanol/ml RLT) and eluted using 16. mu.l nuclease-free water. RNeasy PlusMicro kit (Qiagen, CA) was designed to remove genomic DNA (gDNA), performed according to the manufacturer's instructions, and eluted with 16. mu.l nuclease-free water. For isolation of small RNAs using RNeasyMicro kit or rneasyplus micro kit, miRNA isolation methods were performed according to the manufacturer's instructions. Isolated RNA160 was analyzed on an RNAPico6000 chip (Agilent, CA) using an Agilent BioAnalyzer (Agilent, CA) that can generate an electropherogram of the sample and a corresponding "pseudo-gel".

As shown in fig. 8 and 9, the quality of RNA extracted from urine microvesicles (in terms of yield and integrity) increases when the proteases used to treat the microvesicles are increased before disrupting the microvesicle membrane. In FIG. 8, nucleic acids were extracted from microvesicles in 20ml urine using the modified method above, but RNA extraction was performed using the Qiagen Qiammpmineuteventirusspin kit. The urine samples were concentrated by filtration and eluted with 200. mu.l PBS. Here, we define 1X protease as 0.027 AU; 5X protease is defined as 0.135 AU; 10X protease is defined as 0.27 AU; 25X protease is defined as 0.675 AU; 50X is defined as 1.35 AU. As the protease concentration increased from 5x (a) to 10x (b), 18 and 28SrRNA peaks with greater integrity were observed. Likewise, as shown in fig. 9, at higher protease concentrations, 25x (a) and 50x (b), we observed 18 and 28 srrnas with higher integrity in addition to increased small/miRNA levels. These data indicate that the addition of protease can increase the yield and integrity of 18 and 28SrRNA as well as increase small RNAs and micrornas. We speculate that the action of the protease may be due to its ability to digest away RNases and other inhibitors.

Increasing the number of washing steps of the microbubbles also significantly improves the quality of nucleic acid extracted from the urine microbubbles. This washing step can effectively remove the adverse factors that prevent high quality nucleic acid extraction of urinary microvesicles. Urine samples of 20ml each were used for four sets of nucleic acid extraction tests according to the above method, but with some modifications. For group 1, isolated microvesicles were directly used for nucleic acid extraction without any intervening steps. For group 2, microvesicles were treated with rnase inhibitors prior to nucleic acid extraction. For group 3, the microvesicles were washed prior to nucleic acid extraction, but were not treated with any rnase inhibitor. For group 4, microvesicles were washed and treated with rnase inhibitors prior to nucleic acid extraction. As shown in FIG. 38, for the group 1 test, the quality of the extracted nucleic acids was very poor, with RIN of 3.63. + -. 2.3 and RNA concentration of 101.3. + -. 27.6 pg/. mu.l. For the group 2 test (FIG. 39), the quality of the extracted nucleic acids was approximately similar to that in the group 1 test, where the RIN was 1.83. + -. 2.2 and the RNA concentration was 101.6. + -. 88 pg/. mu.l. For the group 3 test (FIG. 40), the quality of the extracted nucleic acids was significantly improved, with RIN 9.2. + -. 0.0 and RNA concentration 347.7. + -. 97.7 pg/. mu.l. For the 4-group test (FIG. 41), the quality of the extracted nucleic acids was similar to that in group 3, where the RIN was 7.43. + -. 0.2 and the RNA concentration was 346.3. + -. 32.7 pg/. mu.l. These data indicate that, absent a washing step, the quality of the extracts from the microvesicles would be inconsistent with relatively higher variations of those extracts where the microvesicles were washed prior to nucleic acid extraction.

Example 6: application of RNase inhibitor in nucleic acid extraction of serum microvesicles

Using the above improved method, high quality nucleic acid extracts can also be obtained from serum microvesicles. Here, weObtaining serum from melanoma and serum from normal patients, and using RNase-In as RNase inhibitor^TM(Ambion Inc.) treatment of the microvesicle particles by resuspension. In one batch of experiments, we isolated microvesicles from quadruplicate 1ml melanoma serum samples and treated the microvesicles with SUPERAse-In at a final concentration of 1.6 units/. mu.l. The microbubble separation method is ultracentrifugation, and the microbubble particles are treated with DNase at room temperature for 20 minutes. As shown in FIGS. 10-13, the RNA extracts from the four melanoma serum samples were of low and inconsistent quality, RNA yields of 543pg/μ l, 607pg/μ l, 1084pg/μ l, 1090pg/μ l, and RNA integrity was estimated at 28s/18s ratios to be 1.7, 1.8, 1.3 and 0.6, respectively. In another experiment, we isolated microvesicles from duplicate 1ml melanoma serum samples and treated them with SUPERAse-In at a final concentration of 3.2 units/. mu.l. As shown In FIGS. 14 and 15, the amount of RNA extract from two melanoma serum samples treated with 3.2 units of SUPERAse-In/. mu.l was generally better than that of those treated with 1.6 units of SUPERAse-In/. mu.l. The RNA yields of the two melanoma serum samples were 3433pg/μ l and 781pg/μ l, with a 28S/18S ratio of 1.4 and 1.5, respectively.

Further, we tested quadruplicate 1ml normal serum samples at 1.6 units SUPERAse-In/. mu.l and duplicate 1ml normal serum samples at 3.2 units SUPERAse-In/. mu.l. As shown In FIGS. 16 to 19, the RNA extracts were low In quality at 1.6 units of SUPERAse-In/. mu.l, and the RNA yields were 995 pg/. mu.l, 1257 pg/. mu.l, 1027 pg/. mu.l and 1206 pg/. mu.l, and the 28S/18S ratios were 1.3, 1.6 and 1.8, respectively. In contrast, as shown In FIGS. 20 and 21, the RNA extract mass was improved at 3.2 units of SUPERAse-In/. mu.l, RNA yields of 579 pg/. mu.l and 952 pg/. mu.l, and 28S/18S ratios of 1.6 and 2.3.

Example 7: use of an extraction promoter in nucleic acid extracts from biological samples

In examples 3 and 4, our test results show that treatment with an extraction promoter can increase the amount of RNA extract from microvesicles. It is expected that such an extraction promoter will have a similar effect on other biological samples. As shown in fig. 22, the novel nucleic acid extraction method of the present invention will require a step of performing an extraction facilitating operation on a biological sample. Such a method can be exemplified in the nucleic acid extraction experiments envisaged below. The physician prescribes a tumor marker test for the patient. Then 5ml of blood was drawn from the patient. Blood sample 200 is sometimes pre-processed to obtain serum. Then, a boosting operation 210 is performed, for example, by adding a suitable amount of an extraction promoter to the serum and incubating the mixture at 37 ℃ for 30 minutes. Nucleic acids are then extracted from the treated serum using conventional extraction methods as described in detail in example 5 220 and analyzed 230 using an Agilent BioAnalyzer. Such extraction is expected to yield high quality nucleic acids from biological samples.

Nucleic acids from urinary microvesicles as biomarkers

Example 8: contamination of urinary microvesicles with free microvesicles of extracellular non-cellular DNA

We split the urine sample into two 25ml identical samples and isolated the microvesicles from the two subsamples by differential centrifugation as described in detail above. In one subsample, we treated the microvesicles with dnase and extracted the nucleic acids from the treated microvesicles as detailed above. In another subsample, we did not treat the microvesicles with dnase and extract the nucleic acids from the untreated microvesicles. As shown in fig. 23, isolated urine microvesicles were contaminated by free microvesicle extracellular non-cellular DNA. When nucleic acid was extracted using the RNeasymicro kit (fig. 23A and 23B), the results showed that more nucleic acid was seen in the untreated sample (a) than in the treated sample (B), because the peak in (a) was generally higher than the peak in (B).

In another test, we performed a similar test using a serum sample instead of a urine sample. As shown in fig. 24, free extracellular non-cellular DNA also contaminated isolated serum microvesicles. More nucleic acids were seen in the sample (A) without DNase treatment than in the sample (B) treated with DNase, since the peaks in (A) were generally higher than in (B). Similarly, when nucleic acids were extracted using the MirVana kit, as shown in fig. 23(C) and 23(D), the results also show that more nucleic acids were seen in the untreated sample (C) than in the treated sample (D), since the peak in (C) was generally higher than the peak in (D). It is likely that the excess nucleic acid in the untreated sample was from DNase-susceptible "apoptotic bodies" because DNase-susceptible "apoptotic bodies" -like ladders were seen, as shown in the pseudo-gel in FIG. 24C. The amount of free extracellular acellular DNA in urine and serum samples varies from subject to subject, but the size of this DNA is in the range of about 25-1500 base pairs.

Example 9: urinary microvesicles are mostly not contaminated by free microvesicles with extracellular non-cellular RNA

We split the urine sample into two 25ml identical samples and isolated the microvesicles from the two subsamples by differential centrifugation as described in detail above. In one subsample, we treated the microvesicles with rnases and extracted nucleic acids from the treated microvesicles as detailed above. In another subsample, we did not treat the microvesicles with rnases and extracted the nucleic acids from the untreated microvesicles. As shown in FIG. 25, isolated urine microvesicles were hardly contaminated with free microvesicle-external non-cellular RNA. The curve for the sample (a) without rnase treatment largely overlaps the curve for the rnase treated sample (B), indicating the absence of free extra-microvesicle non-cellular RNA associated with the isolated microvesicles. This may be due to ribonucleases present in urine.

Example 10: agilent BioAnalyzer determined similarity of nucleic acid profiles in urine microvesicles and renal cells

We extracted nucleic acids from urine microvesicles and kidney (kidney) tissue and compared their profiles. The method of extracting nucleic acids from urine microvesicles is described in detail in example 5. Rat kidney samples were processed via RNeasyMini kit and RNeasyPlus kit. To determine the amount of small RNA in rat kidney samples, they were also processed through the two kits using miRNA isolation methods according to the manufacturer's instructions.

As shown in figure 26, their profiles (a-kidney, B-microvesicle) are very similar, including the presence and integrity of the 18SrRNA peak and the 28SrRNA peak. Such 18s rrna and 28s rrna peaks were not seen in previously reported serum-derived or cell culture medium-derived microvesicles.

In addition to the similarity of rRNA peaks, urinary microvesicles also contain a small RNA profile similar to that obtained from kidney cells. As shown in FIG. 27, both urinary microvesicles (B) and renal tissue (A) contain small RNAs (about 25-200 base pairs) and share a similar pattern.

These data indicate that, using the novel nucleic acid extraction methods disclosed herein, the profile in urine microvesicles can be used to examine the profile in renal cells from which the microvesicles originate.

Example 11: RNA profile in urinary microvesicles as opposed to whole urine

We found that the RNA profiles in urine microvesicles differ from those in whole urine. We used 75ml duplicate urine samples for testing. The urine was first pretreated at 300g for 10 minutes at 4 ℃, the supernatant centrifuged at 17,000g for 20 minutes at 4 ℃ and the supernatant filtered through a 0.8 μm filter (nitrocellulose membrane filter unit, Nalgene, NY) followed by the procedure detailed in example 5, thus isolating RNA from the urine microbubbles. RNA was isolated from whole urine using a ZR urine RNA isolation kit (ZymoResearch, CA) according to the manufacturer's instructions. To remove DNA from the Zymo processed samples, the eluted RNA was resuspended in 350. mu.l RLT buffer, processed by RNeasy Plus Micro kit using DNase to remove the cognate DNA, and eluted with 16. mu.l nuclease-free water.

As shown in FIG. 28, a large amount of nucleic acid (A) could be isolated using the ZR urine RNA isolation kit without using DNase. However, the profile appears broad, lacking the 18SrRNA and 28SrRNA peaks. Further, the pattern changed significantly when dnase was used (B), indicating that the majority of the extract was DNA in nature. In contrast, as shown in figure 29, the RNA profile of microvesicles from the same urine sample generally differs greatly from the profile from a whole urine extract. In this microvesicle profile, 18SrRNA and 28SrRNA peaks are present. In addition, RNA from microvesicles is more abundant than RNA from whole urine. When the map without dnase treatment (a) was compared with the map with dnase treatment (B), it can be seen that dnase digestion of the extract from microvesicles did not significantly affect rRNA peaks. The RNA profiles from 300g particles (fig. 30) and 17,000g particles (fig. 31) were similar to those from whole urine extracts. Of these two patterns, when the pattern (a) without dnase treatment was compared with the pattern (B) with dnase treatment, it can be seen that the peak was significantly reduced after dnase treatment. These data indicate that DNA is the major species in the extract and that 18SrRNA and 28SrRNA peaks are not detectable. Thus, in conjunction with the data in example 10, it can be seen that the RNA profile from the urine microvesicles more closely resembles the renal cell profile than the profile from whole urine. Further, the integrity of the RNA extract from microvesicles is at least 10 times stronger than that from whole urine.

Example 12: urinary microvesicles containing RNA and DNA

We determined whether urine microbubbles contain RNA, DNA, or both by first treating the particles with rnase and dnase to remove free extracellular non-cellular contamination of the microbubbles, followed by subjecting the nucleic acids within the microbubbles to rnase and/or dnase digestion during column-based nucleic acid isolation. Compared to the nucleic acid pattern (A) without RNase digestion, RNase digestion (B) almost completely destroyed the nucleic acid pattern (FIG. 32). These data indicate that RNA is the most abundant nucleic acid within the microvesicles. As shown in fig. 33, after the rnase-treated sample was subjected to on-column digestion with dnase (B), the peak after only 20s after further dnase digestion inside was reduced compared to the peak before further dnase digestion (a). This reduction demonstrates that a small amount of dnase-digestible material, preferably DNA, is present in the microvesicles.

Example 13: urinary microvesicles containing specific genes encoding mRNA transcripts from various regions of the nephron and ductus venosus

As shown in example 10, the nucleic acid profiles in urine microvesicles and kidney cells were similar as measured by an agilent bioanalyzer. Here we further demonstrate that microvesicles contain mRNA transcripts from various regions of the nephron and ductus effervescens encoding specific genes. Urine microvesicles were isolated from 200ml urine from 2 human subjects (ages 23 to 32 years) and digested with rnase and dnase prior to exosome lysis and RNA extraction, as detailed in example 5. Two rounds of mRNA amplification were performed on the extracted RNA using RiboAmp (molecular devices, CA). For ribose amplification (riboamplification), the samples were incubated at 42 ℃ for 4 hours for the first in-vitro transcription step and at 42 ℃ for 6 hours for the second in-vitro transcription step. The amplified RNA was denatured at 65 ℃ for 5 min and first strand cDNA synthesis was performed as described in the Qiagen Omniscript protocol (Qiagen, CA). GAPDH and β -actin genes were identified in all samples (fig. 34A). We examined 15 transcripts unique to each region of the nephron and ductus efferentes (fig. 34B). These include proteins and receptors associated with various renal diseases, including podocin from the glomerulus, a pinocytotic receptor from the proximal tubule, and aquaporin 2 from the collecting catheter.

For human samples, the PCR primers used were ACTBUTR,

in the forward direction 5'-GAAGTCCCTTGCCATCCTAA-3' of the direction,

reverse ` 5-GCTATCACCTCCCCTGTGTG-3 `;

gapdehx, forward 5'-ACACCCACTCCTCCACCTTT-3',

a reverse direction 5'-TGCTGTAGCCAAATTCGTTG-3';

NPHS2UTR, forward 5'-AACTTGGTTCAGATGTCCCTTT-3',

a reverse direction 5'-CAATGATAGGTGCTTGTAGGAAG-3';

LGALS1EX, forward 5'-GGAAGTGTTGCAGAGGTGTG-3',

a reverse direction 5'-TTGATGGCCTCCAGGTTG-3';

HSPG2UTR，5’-AAGGCAGGACTCACGACTGA-3’，

a reverse direction 5'-ATGGCACTTGAGCTGGATCT-3';

CUBNEX, forward direction 5'-CAGCTCTCCATCCTCTGGAC-3',

a reverse direction 5'-CCGTGCATAATCAGCATGAA-3';

LRP2EX, forward 5'-CAAAATGGAATCTCTTCAAACG-3',

a reverse direction 5'-GTCGCAGCAACACTTTCCTT-3';

AQP1UTR, forward 5'-TTACGCAGGTATTTAGAAGCAGAG-3',

a reverse direction 5'-AGGGAATGGAGAAGAGAGTGTG-3';

CA4UTR, forward 5'-ATGATGGCTCACTTCTGCAC-3',

a reverse direction 5'-TCATGCCTAAAGTCCCACCT-3';

CLCN5EX, forward 5'-GTGCCTGGTTACACACAACG-3',

a reverse direction 5'-AGGATCTTGGTTCGCCATCT-3';

BDKRB1UTR, forward 5'-GTGGTTGCCTTCCTGGTCT-3',

a reverse direction 5'-ATGAAGTCCTCCCAAAAGCA-3';

CALCRUTR, forward 5'-ATTTTGCCACTGCCTTTCAG-3',

a reverse direction 5'-ATTTTCTCTGGGTGCGCTAA-3';

SCNN1DUTR, forward 5'-GCGGTGATGTACCCATGCT-3',

a reverse direction 5'-CTGAGGTGGCTAGGCTTGA-3';

SLC12A3EX, forward 5'-AGAACAGAGTCAAGTCCCTTCG-3',

a reverse direction 5'-TATGGGCAAAGTGATGACGA-3';

AQP2UTR, forward 5'-GCAGTTCCTGGCATCTCTTG-3',

a reverse direction 5'-GCCTTTGTCCTTCCCTAACC-3';

ATP6V1B1EX, Forward 5'-AGGCAGTAGTTGGGGAGGAG-3',

a reverse direction 5'-CGAGCGGTTCTCGTAGGG-3';

SLC12A1EX, forward 5'-CAGATGCAGAACTGGAAGCA-3',

and reverse direction 5'-GGAAGGCTCAGGACAATGAG-3'. "UTR" refers to a primer designed into the UTR, and "EX" refers to a primer designed across an exon. The PCR protocol was 94 ℃ for 5 minutes; 94 ℃ for 40 s; 30s at 55 ℃; 1 minute at 65 ℃ for 30 cycles; and 68 ℃ for 4 minutes. For mouse samples, the primers used were: AQP 2: forward 5'-GCCACCTCCTTGGGATCTATT-3', reverse 5'-TCATCAAACTTGCCAGTGACAAC-3';

V-ATPaseB1 subunit: in the forward direction 5'-CTGGCACTGACCACGGCTGAG-3' of the direction,

and reverse direction 5'-CCAGCCTGTGACTGAGCCCTG-3'. The PCR protocol was 94 ℃ for 5 minutes; 30 cycles of 94 ℃ for 40s, 55 ℃ for 30s, and 65 ℃ for 1 min; and 68 ℃ for 4 minutes.

As shown in fig. 34, panel a, RiboAmp-amplified mRNA transcripts of β -actin and GAPDH were readily detected in a pseudo-gel of 4 human subject urinary microvesicles generated by BioAnalyzer. For clarity, six regions of nephrons and collecting catheters are shown in fig. 34, group B. The following transcripts in the six regions were also readily detected by RT-PCR analysis of RiboAmpedmRNA from urinary exosomes: region 1 glomeruli: NPHS2-podocin, LGALS 1-galectin-1, and HSPG 2-heparan sulfate proteoglycan (FIG. 35); region 2 proximal tubule: CUBN-gulp receptor, LRP 2-megaprotein, AQP 1-aquaporin 1, CA 4-carbonic anhydrase 4, and CLCN 5-chloride channel protein 5 (fig. 35); zone 3 fine descending branches: BDKRB 1-bradykinin B1 receptor (fig. 36); zone 4 thick ascending branches of medulla: the CALCR-calcitonin receptor, and SCNN 1D-amiloride sensitive sodium channel subunit (FIG. 36); region 5 distal convoluted tubule: SLC12a 3-thiazide-sensitive sodium chloride cotransporter (fig. 36); region 6 manifold catheters: AQP 2-aquaporin 2, ATP6V1B1-vATPaseB1 subunit, and SLC12A 1-kidney specific Na-K-C1 symporter (FIG. 36).

Thus, mRNA transcripts from all renal regions examined can be identified, indicating that all regions of the nephron and ductus effervesces containing mRNA are released, and that microvesicles can be a new non-invasive source of nucleic acid biomarkers of renal disease.

Example 14: some mRNA transcripts in urinary microvesicles are specific to renal cells

If the disease-causing renal genes are examined non-invasively using nucleic acids in the microvesicles, the transcripts in the microvesicles are specific to renal cells. Here, we demonstrate that mRNA transcripts are specific for kidney cells. We used knock-out mice lacking the V-ATPase B1 subunit. Deficiency of V-ATPaseB1 can lead to renal acidosis in mice (FinbergKE, WagnerCA, BaileyMA, et al, the Bl-Subunitofthe (+) ATPaseseisrequiredformimaxiliaryyidification. ProcNatl Acad Sci USA 102: 13616-13621.2005).

All animal experiments were performed according to the animal ethical guidelines approved by massachusetts general hospital. A V-ATPaseB1 subunit knock-out animal has been described (FinbergKE, WagnerCA, BaileyMA, et al. the Bl-SubunitoftheH (+) ATPaseISREQUIRE DEFORMALIMAXINAL ARYACIFIcation. ProcNatlAcadSciUSA 102: 13616-. To collect urine, animals in 2 groups (n-4 animals per group) were housed in metabolic cages for 72 hours (sufficient RNA could also be obtained by housing one animal per cage) and urine was collected for microvesicle isolation and analysis as described above for human urine. To extract the kidneys, animals were anesthetized with sodium pentobarbital (Nembutal) (abbott laboratories, IL) (65mg/kg body weight, i.p., injection), and the kidneys were immediately removed and frozen in liquid nitrogen. The frozen kidneys were crushed using a pestle and mortar in a liquid nitrogen bath, resuspended in RNAlater (Qiagen, CA) and stored in 1ml aliquots at-80 ℃. To extract RNA, aliquots were thawed on ice and 50. mu.l lysed in 350. mu.l RLT buffer (with 10. mu.l. beta. -mercaptoethanol/ml RLT). Mouse kidney samples were processed by RNeasyMini kit (Qiagen, CA) including DNA digestion steps.

To perform real-time PCR analysis, RNA extracted from mouse urinary microvesicles was denatured at 65 ℃ for 5 minutes and first strand cDNA synthesis was performed as described in the Qiagen sensiscript protocol (Qiagen, MD). To perform the Sensiscript reverse transcription, oligo-dT primers were used at a final concentration of 1. mu.M (applied biosystems, Calif.). The resulting cDNA was used in a taqman preamp mastermix kit according to the manufacturing guidelines (applied biosystems, CA) using 14 pre-amplification cycles. The pre-amplified product was then diluted with 1XTE buffer (Promega, Wis.). The resulting cDNA was then used as a real-time PCR template according to the Taqman amplification guide (applied biosystems, Calif.). Mouse renal RNA concentrations were measured on a SmartSpec3000(Bio-Rad, CA) and all samples were diluted to 90 ng/. mu.l. Mouse kidney RNA was denatured at 65 ℃ for 5 min and first strand cDNA synthesis was performed as described in the Qiagen Omniscript protocol (Qiagen, MD). In performing Omniscript reverse transcription, oligo-dT primers were used at a final concentration of 1. mu.M (applied biosystems, Calif.), followed by 1. mu.l of the resulting cDNA per well in a subsequent real-time PCR reaction. The real-time PCR reaction was performed on ABI7300RealTimePCRSystem (applied biosystems, CA) using the TaqMan Gene expression MasterMixandExpression assays (MouseGAPDPartNumber4352339E and mouse Atp6v1b2 assay idMm00431996_ mH).

We extracted RNA from kidney tissue and urinary microvesicles of knockout mice. We examined the expression of V-ATPase B1 subunit and aquaporin 2(AQP2) mRNA using RT-PCR. As shown in FIG. 37, panel A, no V-ATPaseB1 subunit transcript was detected in both kidney and urine microbubble samples from double mutant mice (B1-/-), consistent with the fact that the V-ATPaseB1 subunit gene was knocked out in these mice. In contrast, V-ATPase B1 subunit transcripts were present in kidney and microbubble samples from wild type mice (B1/+/+). AQP2mRNA was readily detected in kidney and microvesicle samples of B1 knockout or wild type mice, which was expected because V-atpase B1 subunit deletion did not affect expression of AQP 2. Further, as shown in fig. 37, panel B, the expression level of V-atpase B2 subunit in B1 knockout mouse microvesicles was not statistically different from that in wild mouse microvesicles. The expression level of V-ATPaseB2 subunit in kidney cells of knockout mice is the same as that in kidney cells of wild-type mice. Thus, transcripts present in kidney cells can be detected in urine microvesicles secreted by the kidney cells, whereas transcripts not present in kidney cells cannot be detected in urine microvesicles secreted by the kidney cells. Thus, the transcripts in the urinary microvesicles are specific to kidney cells and are non-invasive biomarkers of kidney cell transcripts.

Example 15: urinary microvesicles containing non-coding RNA transcripts

Urine microvesicles were isolated and nucleic acids were extracted according to the above method described in detail in example 5. We performed deep sequencing of the urinary microvesicle RNA and found that random regions exhibiting extreme transcription (extremtrancription) were present on some chromosomes. When the number of transcripts is plotted against position on the chromosome, these transcripts appear in the form of "spikes". These transcripts are more abundantly expressed than well known endogenous markers such as GAPDH or actin, and are generally located in non-coding regions of the chromosome. The relatively high level expression of these spike sequences reveals that these sequences may also play an important role in chromosome activation and cell regulation.

We identified 29 regions where there were more than 500 peaks. The 29 regions are shown in FIG. 42, corresponding to SEQ ID NO. 1-29. The spikes in these 29 regions plotted are shown in fig. 45-73. PCR analysis of the most highly expressed spike transcript sequences demonstrated that they were indeed present in human urine microvesicles and human kidney cells, indicating that these sequences are not artifacts of deep sequencing. Primers used to amplify 10 such peak-rich regions are shown in fig. 43. PCR was performed according to the following protocol: initial denaturation at 95 ℃ for 8 min; 30 cycles consisting of three steps of denaturation at 95 ℃ for 40 seconds, annealing at 55 ℃ for 30 seconds and extension at 65 ℃ for 1 minute; final extension at 68 ℃ for 5 min; and kept at 4 ℃ before analyzing the reaction with a BioAnalyzer. As shown in fig. 44, using the template, amplification of each of these 10 regions in both human urine microvesicles (a) and human kidney cells (B) gave positive results, indicating that these spike transcripts were indeed present in microvesicles and human kidney cells.

These abundant spike transcripts can be used to assess nucleic acid extract quality from biological samples. For example, the amount of any of the spike transcripts can be used to assess the quality of nucleic acids from urinary microvesicles in place of common markers such as GAPDH or ACTIN polynucleotide molecules. The amount of GAPDH or ACTINRNA in urine microvesicles is so low that an additional amplification step such as RiboAMP is required to measure their amount. In contrast, the amount of any one of the spiking transcripts is so high that no additional amplification step is required. Thus, the use of these spike transcripts may make the assessment of nucleic acid extract quality more efficient and simpler. Thus, another aspect of the invention described herein is a novel method for assessing the quality of nucleic acid extracts from biological samples, such as human urine samples. The method can be completed by the following operations: by extracting nucleic acids from a biological sample, measuring the amount of any one of the spike transcripts, and comparing that amount to a standard that has been determined for the particular biological sample. Such criteria can be, for example, the average amount of such peak transcripts extracted from 10 normal human urine samples, as can be established by experienced biotechnological professionals.

Although the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations and changes to the described embodiments are possible without departing from the scope of the invention, which is defined by the appended claims. Accordingly, it is intended that the invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims, and equivalents thereof.

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Claims (9)

1. A method of obtaining mRNA from a urine sample, comprising the steps of:

(a) obtaining a urine sample;

(b) obtaining a microvesicle fraction from the urine sample by performing the following steps:

(i) filtering the urine sample through a 0.8 μm filter,

(ii) the microvesicle fraction was isolated using ultrafiltration concentration,

(iii) washing the isolated microvesicle fraction;

(c) performing an extraction-promoting operation on the microvesicle fraction from the urine sample, wherein the promoting operation comprises adding an rnase inhibitor to the isolated microvesicle fraction;

(d) extracting mRNA from the microvesicle fraction; and

(e) comparing the amount of the mRNA to a standard.

2. The method of claim 1, wherein the rnase inhibitor is present at a concentration greater than 0.027 AU.

3. The method of claim 1 or 2, wherein the rnase inhibitor is a protease.

4. The method of claim 1, further comprising treating the microvesicle fraction with a dnase enzyme to remove all or substantially all of any DNA located outside or on the surface of microvesicles in the sample; extracting mRNA from the sample; and analyzing the extracted mRNA.

5. The method of claim 1, wherein the nucleic acid yield from a 20ml urine sample is greater than or equal to 50 pg/ml.

6. The method of claim 1, wherein the concentration of the rnase inhibitor is greater than or equal to 0.135 AU.

7. The method of claim 1, wherein the concentration of the rnase inhibitor is greater than or equal to 0.27AU concentration.

8. The method of claim 1, wherein the concentration of the rnase inhibitor is greater than or equal to 0.675AU concentration.

9. The method of claim 1, wherein the concentration of the rnase inhibitor is greater than or equal to 1.35AU concentration.