WO2008157316A1

WO2008157316A1 - Improved method for rna isolation

Info

Publication number: WO2008157316A1
Application number: PCT/US2008/066866
Authority: WO
Inventors: Kristen Drescher; Baha Abdalhamid
Original assignee: Creighton University
Current assignee: Creighton University
Priority date: 2007-06-14
Filing date: 2008-06-13
Publication date: 2008-12-24
Anticipated expiration: 2009-12-14

Abstract

The present invention provides methods for isolating RNA from a biological sample, preferably an RNAse-rich biological sample. The method includes mixing a biological sample with an extraction reagent to form a homogenate, extracting the RNA in the homogenate with an organic solvent, and precipitating RNA from the aqueous phase. In some aspects the biological sample and the extraction reagent are combined at a ratio of less than 50 mg of the biological sample to 1 ml extraction reagent, preferably, no greater than 25 mg of the biological sample to 1 ml extraction reagent. In some aspects, the organic solvent includes phenol and chloroform in a ratio of between 4 parts phenol to 1 part chloroform (4: 1) and 6 parts phenol to 1 part chloroform (6: 1).

Description

IMPROVED METHOD FOR RNA ISOLATION

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Serial No. 60/934,517, filed June 14, 2007, which is incorporated by reference herein.

GOVERNMENT FUNDING

The present invention was funded by the State of Nebraska Legislative Bill LB 692 (Nebraska Tobacco Settlement Biomedical Research Program) allocation to Creighton University. The State of Nebraska has certain rights in this invention.

BACKGROUND Analyzing gene expression by Northern blot, ribonuclease (RNAse)

Protection Assays, reverse transcriptase-polymerase chain reaction (RT-PCR), Real-Time RT-PCR, and microarray requires the isolation of intact, high quality RNA from cells. One of the main problems encountered in obtaining intact RNA is the ubiquitous presence of RNAses in cells. These ribonucleases are extremely stable and require inactivation in order to prevent degradation of the RNA sample. Use of poor quality or partially degraded RNA may lead to erroneous results when these samples are used in gene expression studies. Certain tissues, such as pancreas and spleen, contain high levels of RNAses. RNA isolation protocols have become simplified over the past 25 years, particularly with the advent of commercially-available RNA isolation kits.

Despite the availability of kits that permit the isolation of high-quality RNA in a short amount of time, most RNA isolation kits are not particularly useful with tissues containing high levels of RNAses (spleen, pancreas, placenta, etc.). Because of this, the most effective means of isolating high quality RNA from these particular tissues tend to be more laborious and time-consuming. SUMMARY OF THE INVENTION

The present invention represents an advance in the art of obtaining RNA from biological sources. Intact mRNA that can be used in experiments, such as production of cDNA, is notoriously difficult to obtain from tissues. Partial or complete degradation of transcripts can lead to compromised data, particularly if there is preferential or biased degradation of certain transcripts. Methods are available for obtaining mRNA from some sources; however, it remains difficult to obtain mRNA from certain ribonuclease (RNase)-rich tissues in sufficient quantity and of sufficient quality. The methods of the present invention are easy to perform, highly reproducible, and yield highly intact isolated RNA from biological samples, including biological samples rich in RNase. The mRNA obtained using the methods described herein can be used in a variety of methods such as microarray and Real -Time RT-PCR.

The present invention provides a method for isolating RNA from a biological sample. The method includes mixing a biological sample with an extraction reagent to form a homogenate, extracting the RNA in the homogenate with an organic solvent, and precipitating RNA from the aqueous phase. The biological sample may be RNase-rich, such as, but not limited to, pancreas and spleen. Preferably, the biological sample includes at least 0.5 milligram (mg) RNases per gram of biological sample, at least 1 mg RNases per gram of biological sample, at least 1.2 mg RNases per gram of biological sample, or at least 1.4 mg RNases per gram of biological sample. The biological sample and the extraction reagent may be combined at a ratio of less than 50 mg of the biological sample to 1 ml extraction reagent, preferably, no greater than 25 mg of the biological sample to 1 ml extraction reagent. The extraction reagent may include a guanidinium compound, such as guanidine isothiocyanate, and phenol. Preferred examples of the extraction reagent are available under the tradename Trizol® and Tri Reagent®. The extraction reagent may include a reducing agent, such as β-mercaptoethanol, which may be present in the extraction reagent at 1% (vol/vol).

The RNA in the homogenate may be extracted with an organic solvent. An organic solvent used in the extraction may include phenol and chloroform in a ratio of between 4 parts phenol to 1 part chloroform (4:1) and 6 parts phenol to 1 part chloroform (6:1). The phenol may have a pH between 4.1 and 4.5. An organic solvent used in the extraction may be chloroform as the organic solvent. An organic solvent used in the extraction may be chloroform and isoamyl alcohol. The precipitating may include adding an alcohol and recovering the RNA.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances.

However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention. The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Gel electrophoresis for RNA isolated from pancreas using Trizol or modified Trizol methodology. Lane 1 - RNA sample treated with Trizol, β-mercaptoethanol, Phenol (pH4.3): chloroform (125:24), and

Proteinase K treatment. Lane 2- RNA sample was treated as in lane 1 without proteinase K treatment. Lane 3- RNA sample was treated with Trizol.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes extraction reagents. An extraction reagent may include a guanidinium compound, phenol, or, preferably, a combination thereof. The guanidinium compound may be, for instance, an acid guanidinium, or a salt thereof, such as guanidinium thiocyanate, or guanidinium hydrochloride. Another example of a guanidinium compound is guanidine isothiocyanate. The amount of guanidinium compound may be at least 0.5 M, or at least 0.75 M, based on the total volume of the extraction reagent, and may be no greater than 1 M, or no greater than 2 M, no greater than 3 M, or no greater than 4 M, based on the total volume of the extraction reagent. The amount of phenol may be at least 30%, or at least 35% by volume based on the total volume of the extraction reagent, and may be no greater than 40%, or no greater than 50% by volume based on the total volume of the extraction reagent.

The extraction reagent of the present invention may include one or more additional thiocyanate components including, but not limited to, ammonium thiocyanate or sodium thiocyanate. The amount of thiocyanate compound may be at least 0.1M, at least 0.2M, or at least 0.3, based on the total volume of the extraction reagent, and may be no greater than 0.5 M, or no greater than 0.6 M, based on the total volume of the extraction reagent.

The extraction reagent may be buffered to maintain a pH of, for instance, between 4 and 6. The extraction reagent may include a buffering compound, such as, but not limited to, sodium acetate or sodium citrate, in an amount sufficient to maintain the pH of the solution in the range of between pH 4 and pH 6. The extraction reagent may include an additional solubilizer for maintaining the phenol in solution. An example of a suitable solubilizer is glycerol. The solubilizer, such as glycerol, may be at least 3%, or at least 4% by volume based on the total volume of the extraction reagent, and may be no greater than 7%, no greater than 8%, no greater than 9%, or no greater than 10% by volume based on the total volume of the extraction reagent. In a preferred embodiment of the extraction reagent, the concentration of guanidinium thiocyanate is 0.8M, the concentration of ammonium thiocyanate is 0.4M, the sodium acetate buffer is present in a concentration of 0.1 M, based on the total volume of the solvent solution, whereby the solution has a pH of 5.0. Additionally, the solvent solution includes 5% by volume glycerol and 38% by volume phenol. Examples of useful extraction reagents are disclosed in Chomczynski (U.S. Patent 5,346,994), and Chomczynski and Sacchi (Anal. Biochem, 1987, 162: 156-159). Preferred extraction reagents are commercially available under the tradename Trizol® reagent (Invitrogen, Carlsbad, CA) and Tri Reagent® (Molecular Research Center, Inc., Cincinnati, OH). The extraction reagent may also include a reducing agent. Examples of useful reducing agents include, but are not limited to, β-mercaptoethanol, dithiothreitol (DTT), other mercaptans, or a combination thereof. The reducing agent may be present in various concentrations. For instance, when the reducing agent is β-mercaptoethanol, it may be present from at least 0.5% (vol/vol) to at least 1% (vol/vol). In some aspects, β-mercaptoethanol may be present at 1% (vol/vol). While concentrations of β-mercaptoethanol at concentrations greater than 1 % (vol/vol) may be used, it may be necessary to extract the sample additional times with an organic solvent. Extraction with organic solvents is described below. Extraction with organic solvents is described below. In some aspects, DTT may be present at between 0.1 M and 0.3M, preferably, 0.2 M. The present invention includes methods for isolating RNA. As used herein, "isolated" refers to a molecule, such as an RNA molecule, that has been removed from its natural environment. Preferably, an RNA molecule is purified, i.e., essentially free from any other polynucleotides, polypeptides, and associated cellular products or other impurities. A "purified" RNA molecule is one that is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. Typically, the components used in the methods, including the reagents and labware, such as test tubes, are RNAse free. Methods for treating components, including reagents and labware, to remove and/or inactivate RNAse, are known in the art and used routinely. Such methods include treatment with diethylpyrocarbonate, autoclaving, and the like.

A method of the present invention may include mixing a biological sample with an extraction reagent to form a homogenate. As used herein, a "biological sample" refers to a sample of cells, such as cultured cells or cells present in a tissue. Common sources of a biological sample include, but are not limited to, blood, excised tissues such as whole organs and biopsies, and cultured animal or plant cells lines. The biological sample can be obtained from any animal, such as a mammal (including, for instance, mouse, rat, or human), or an avian (including, for instance, chicken), or from a plant. The biological sample is preferably an RNAse-rich biological sample. As used herein, an "RNase-rich" biological sample is a biological sample with a high level of ribonuclease present. Preferably, an RNase-rich biological sample includes at least 0.5 milligram (mg) RNases per gram of biological sample, at least 1 mg RNases per gram of biological sample, at least 1.2 mg RNases per gram of biological sample, or at least 1.4 mg RNases per gram of biological sample. Methods for measuring the concentration of RNAses in a biological sample are routine and known in the art. Examples of RNase-rich biological samples include, for instance, pancreas, spleen, intestine, placenta, liver, leukocytes, amoeba, and plant material such as, but not limited to, tomato fruit.

The amount of extraction reagent combined with the biological sample can vary. The biological sample and extraction reagent may be combined in a ratio of less than 50 mg biological sample to 1 milliliter (ml) extraction reagent (less than 50 mg: 1 ml), less than 40 mg biological sample to 1 ml extraction reagent (less than 40 mg:l ml), or less than 30 mg biological sample to 1 ml extraction reagent (less than 30 mg:l ml). The biological sample and extraction reagent may be combined in a ratio of greater than 10 mg biological sample to 1 ml extraction reagent (greater than 10 mg: 1 ml), or greater than 20 mg biological sample to 1 ml extraction reagent (greater than 20 mg:l ml). A preferred ratio is 25 mg biological sample to 1 ml extraction reagent (25 mg: 1 ml). In this aspect volumes of extraction reagent can be greater or less than 1 ml, provided the ratio is as described herein. Typically, the weight of the biological sample is fresh weight.

The biological sample and extraction reagent are mixed to form a homogenate. Preferably, the mixing is done rapidly to promote contact of the RNA-degrading enzymes of the cells with the extraction reagent. Generally, a homogenizer is used. The resulting homogenate may be treated to remove large particulate matter. For instance, the homogenate may be filtered or subjected to centrifugation. Typically, the homogenate contains other biological components such as proteins, lipids, carbohydrates, and deoxyribonucleic acids. These and other biological components can be removed by extracting the RNA. Extracting the RNA may include adding to the homogenate a compound, such as one or more organic solvents, that can act to partition the unwanted biological components into the organic phase and away from the RNA present in the aqueous phase. Accordingly, the method may include adding a compound, preferably, an organic solvent, to the homogenate. Examples of organic solvents that can be used include, but are not limited to, phenol, chloroform, and the like, and combinations thereof. Typically, an organic solvent is added, the mixture of homogenate and organic solvent is mixed, and the resulting aqueous phase removed and transferred to another container. The method may include the single addition of an organic solvent, followed by mixing and removal of the aqueous phase. Optionally, the aqueous phase may be extracted one or more additional times. Preferred organic solvents include, but are not limited to, phenol, a combination of phenol and chloroform, and a combination of chloroform and isoamyl alcohol. The phenol used as an organic solvent alone or in combination with another organic solvent may be at a pH of between 4.1 and 4.5, preferably, pH 4.3. Methods for bringing the pH of a phenol solution to between 4.1 and 4.5 are know in the art and are routine. When phenol and chloroform are used together, the phenol and chloroform can be used at a ratio of between 1 part phenol to 1 part chloroform (1 :1) and 6 parts phenol to 1 part chloroform (6:1), preferably between 4 parts phenol to 1 part chloroform (4:1) and 6 parts chloroform to 1 part chloroform (6:1), more preferably 125 parts phenol to 24 part chloroform (125:24). In some aspects, the ratio of phenol to choroform is greater than 1 part phenol to 1 part chloroform (greater than 1 :1). As used herein, a ratio of two liquids refers to the amount of one liquid relative to the amount of the second liquid. For instance, a 1 : 1 ratio of two liquids means the solution is made up of equal of volumes of each liquid, and a 6:1 ratio of two liquids means the solution is made up of 6 parts of one liquid and 1 part of the second liquid. Chloroform may be used as an organic solvent alone, or may be combined with isoamyl acohol. The chloroform and isoamyl alcohol may be combined at a ratio of between 25 parts chloroform to 1 part isoamyl alcohol (25:1) and 23 parts chloroform to 1 part isoamyl alcohol (23:1), preferably, a ratio of 24 parts chloroform to 1 part isoamyl alcohol (24:1).

Various combinations of extractions with organic solvents can be used in the method of the present invention. Typically, the first organic solvent is added to the homogenate, the mixture is vigorously shaken, and then the mixture is allowed to incubate for several minutes and, optionally, centrifuged. The resulting aqueous phase is carefully transferred to a new tube, and, if an additional extraction is to be done, the process of adding organic solvent, shaking, incubating, and centrifuging is repeated. In one aspect, the extracting includes extracting with chloroform, extracting with phenol : chloroform, and extracting with chloroform :isoamyl alcohol. Organic solvents may be used in any order.

Optionally, the RNA may be precipitated after the extracting. Many methods for precipitating RNA are known in the art and are used routinely. Preferably, an alcohol is used. For instance, isopropanol or ethanol can be used. After precipitation the resulting pellet can be suspended in an appropriate buffer, such as one treated with diethylpyrocarbonate (DEPC), and the RNA can be used immediately or stored until needed.

Optionally, the solution containing RNA can be further treated with one or more proteinases. This may be done with the RNA either before or after it is precipitated and suspended. Many proteinases are available and can be used. In one aspect, proteinase K is used. Proteinase K is a well known proteinase and is commercially available. For instance, 0.5 microgram (μg) per microliter (μl) of proteinase K may be added to the RNA and then incubated for an hour at a temperature that permits the proteinase K to be active, for instance, 55°C. The mixture is then extracted to remove the proteinase K, for instance by extracting with an organic solvent, such as phenol :chloroform, and then precipitated. In some aspects, the precipitation is accomplished by the addition of sodium acetate with ethanol. After precipitation the resulting pellet can be suspended in an appropriate buffer, such as one treated with DEPC, the RNA can be used immediately or stored until needed.

The purity of the RNA can be determined by measuring the absorbance at 260 nanometers (nm) and 280 nm of the isolated RNA. Preferably, the

A260/A280 is between 1.9 and 2.1. The quality of the RNA obtained using the methods described herein can be measured by gel electorphoresis. For instance, when an RNA sample obtained as described herein is resolved using gel electrophoresis and the resolved RNA is visualized, the bands corresponding to 28 and 18S rRNA should visible, and the presence of these two bands in a ratio of approximately 2:1 generally indicates intact RNA. The absence of one or more of these bands can indicate that the RNA is degraded.

The present invention also provides a kit for isolating, preferably purifying, RNA. The kit includes an extraction reagent, one or more organic solvents, RNase-free buffer, or a combination thereof, in a suitable packaging material in an amount sufficient for at least one isolation. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. Instructions for use of the packaged polypeptide or primer pair are also typically included. As used herein, the phrase "packaging material" refers to one or more physical structures, for instance, separate containers, used to house the contents of the kit. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the kit materials can be used for RNA isolation. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to isolate RNA. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an extraction reagent, one or more organic solvents, RNase-free buffer, or a combination thereof. Thus, for example, a package can include plastic vials used to contain - quantities of an extraction reagent, one or more organic solvents, RNase-free buffer, or a combination thereof. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one isolation method parameter, such as the relative amounts of reagent and biological sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, and the like.

Example 1

Isolation of RNA from RNase-Rich Tissues

TRIZOL (Invitrogen; Carlsbad, CA) solution was modified by supplementing with 1% (vol/vol) β-mercaptoethanol (Sigma, St. Louis, MO) and was added to fresh pancreatic tissue at a ratio of 1 ml modified TRIZOL for every 25 mg of pancreatic tissue. From this point on all reagents, components, and surfaces that came into contact with the sample were RNase-free. Tissue was ground using an electric homogenizer for 10-15 seconds until the tissue was completely homogenized. The resulting homogenate was separated into 1 ml aliquots in 1.5 ml microfuge tubes. Insoluble materials were removed from the homogenate by centrifugation in a microfuge at 12,000 x g for 10 minutes at 4°C. The cleared homogenate was collected, transferred to a clean microfuge tube, and incubated for 5 minutes at room temperature (RT).

A volume of 200 μl of chloroform was added to each tube of cleared homogenate solution. The samples were mixed for 30 seconds by vigorous shaking. Samples were incubated for 3 minutes at RT and were centrifuged in a microfuge at 12,000 x g for 15 minutes at 4°C. The aqueous phase (approximately 350 μl) was carefully collected and transferred to a clean microfuge tube. A solution of phenol, adjusted to pH 4.3 with citric acid (Sigma), and chloroform (Sigma) was prepared at a phenol: chloroform ratio of 125:24. An equal volume of the solution was added to each sample. The samples were mixed for 30 seconds by vigorous shaking and incubated on ice for 3 minutes. Samples were centrifuged in a micro fuge at 12000 x g for 15 minutes at 4°C. Following centrifugation, 200 μl of aqueous phase were carefully collected and transferred to a clean microfuge tube.

A volume of 200 μl of chloroform: isoamyl alcohol (Sigma) at a ratio of 24:1 was added to each sample. Samples were mixed for 30 seconds by vigorous shaking. Samples were centrifuged in a microfuge at 12,000 x g for 15 minutes at 4°C. A volume of 150 μl of aqueous phase was carefully collected and transferred to a clean microfuge tube.

RNA was precipitated from each sample by adding 0.5 ml of isopropanol (Sigma) and incubating for 10 minutes at RT. Alternatively, samples can be stored at -80 ⁰C until ready to be used. RNA was pelleted by centrifugation in a microfuge at 12,000 x g for 10 minutes at 4 ⁰C. The supernatant was carefully removed and discarded. The pellet was washed with 70% ethanol and centrifuged in a microfuge at 7500 x g for 5 minutes. The supernatant was carefully and completely removed and the pellet was allowed to air dry for 5 minutes at RT. The pellet was resuspended in 50 μl of Ix diethylpyrocarbonate (DEPC)-treated TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). High quality RNA was routinely obtained at this step.

Performing a final treatment with proteinase K after resuspension in 50 μl of Ix DEPC-treated TE is an optional step. A volume of 1 μl of proteinase K (20 mg/ml) was used in a 40 μl reaction bringing the final concentration of proteinase K (Qiagen, Valencia, CA) to 0.5 μg/μl in each sample. Samples were incubated for 1 hour at 55°C. The total sample volume was adjusted to 200 μl using Ix DEPC-treated TE buffer. RNA was extracted as described above with an equal volume of phenol (pH 4.3):chloroform:isoamyl alcohol (Sigma, St.

Louis, MO) at a ratio of 25:24:1. A volume of 180 μl of the aqueous phase was transferred to a clean microfuge tube. RNA was precipitated from the solution by the addition of 1/10 volume 3M sodium acetate at pH 5.2 and 2.5-3 volumes of 100% ethanol. Other acetates should work in a similar manner, such as ammonium acetate with final concentration of 2.5 M. Samples were mixed well, incubated for at least 2 hours at -80 ⁰C, and RNA was pelleted by centrifugation in a microfuge at 12,000 x g for 10 minutes at 4 ⁰C. The supernatant was carefully removed and discarded. The pellet was washed with 70% ethanol and centrifuged in a microfuge at 7500 xg for 5 minutes. The supernatant was carefully and completely removed and the pellet was allowed to air dry for 5 minutes at RT. The pellet was resuspended in 50 μl of Ix DEPC-treated TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The absorbance at 260 nanometers (nm) and 280 nm of the isolated

RNA was determined using a GeneQuant pro (Amersham Bioscience, Piscataway, NJ). The A260/A280 was routinely 2.0 to 2.1, thus indicating that high quality RNA resulted from this procedure.

RNA obtained as described herein and obtained using a normal Trizol methodology was resolved on a 1 % agarose gel and stained with ethidium bromide as shown in Figure 1. RNA was isolated from pancreas using standard methods (lane 3) or the methods described herein (lanes 1 and 2). Samples were treated with (lane 1) or without (lane 2) proteinase K. In lanes 1 and 2, 2 bands corresponding to 28 and 18 S rRNA can be clearly seen on the gel with a ratio of approximately 2:1 which indicates intact RNA. However, 28S rRNA was not observed in the sample treated only with Trizol (lane 3) which indicates that RNA is degraded. These data suggest that the RNA obtained using the methods described herein is of higher quality than that which was obtained using the standard method when isolating RNA from RNase-rich tissues. In addition, these data indicate that there is no difference in RNA quality between samples treated in the presence or absence of proteinase K (lane 1 and lane 2, respectively).

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A method for isolating RNA from a biological sample comprising: mixing a biological sample with an extraction reagent to form a homogenate, wherein the biological sample and the extraction reagent are combined at a ratio of less than 50 mg of the biological sample to 1 ml extraction reagent, and wherein the extraction reagent comprises a reducing agent; extracting the RNA with an organic solvent, wherein the organic solvent comprises phenol and chloroform in a ratio of between 4 parts phenol to 1 part chloroform (4:1) and 6 parts phenol to 1 part chloroform (6:1); and precipitating RNA from the aqueous phase.

2. The method of claim 1 wherein the biological sample is an RNase-rich tissue.

3. The method of claim 2 wherein the RNase-rich tissue is pancreas or spleen.

4. The method of claim 1 wherein the extraction reagent comprises a guanidinium compound and phenol.

5. The method of claim 4 wherein the guanidinium compound is guanidine isothiocyanate.

6. The method of claim 1 wherein the extraction reagent is sold under the tradename Trizol® or Tri Reagent®.

7. The method of claim 1 wherein the precipitating comprises adding an alcohol and recovering the RNA.

8. The method of claim 1 wherein no greater than 25 mg of the biological sample is mixed with 1 ml extraction reagent.

9. The method of claim 1 wherein the reducing agent is β-mercaptoethanol.

10. The method of claim 9 wherein β-mercaptoethanol is present in the extraction reagent at 1% (vol/vol).

1 1. The method of claim 1 wherein the phenol has a pH between 4.1 and 4.5.

12. The method of claim 1 wherein the extracting comprises chloroform as the organic solvent.

13. The method of claim 1 wherein the extracting comprises chloroform and isoamyl alcohol as the organic solvent.

14. The method of claim 1 wherein the extracting further comprises extracting with chloroform and extracting with chloroform and isoamyl alcohol.

15. A method for isolating RNA from a biological sample comprising: mixing a biological sample with an extraction reagent to form a homogenate, wherein the extraction reagent comprises a reducing agent; extracting the RNA with an organic solvent, wherein the organic solvent comprises phenol and chloroform in a ratio of between 4 parts phenol to 1 part chloroform (4:1) and 6 parts phenol to 1 part chloroform (6:1); and precipitating RNA from the aqueous phase.

16. The method of claim 15 wherein the biological sample is an RNase-rich tissue.

17. The method of claim 16 wherein the RNase-rich tissue is pancreas or spleen.

18. The method of claim 15 wherein the extraction reagent comprises a guanidinium compound and phenol.

19. The method of claim 18 wherein the guanidinium compound is guanidine isothiocyanate.

20. The method of claim 15 wherein the extraction reagent is sold under the tradename Trizol® or Tri Reagent®.

21. The method of claim 15 wherein the precipitating comprises adding an alcohol and recovering the RNA.

22. The method of claim 15 wherein no greater than 25 mg of the biological sample is mixed with 1 ml extraction reagent.

23. The method of claim 15 wherein the reducing agent is β- mercaptoethanol .

24. The method of claim 23 wherein β-mercaptoethanol is present in the extraction reagent at 1% (vol/vol).

25. The method of claim 15 wherein the phenol has a pH between 4.1 and 4.5.

26. The method of claim 15 wherein the extracting comprises chloroform as the organic solvent.

27. The method of claim 15 wherein the extracting comprises chloroform:isoamyl alcohol as the organic solvent.

28. The method of claim 15 wherein the extracting further comprises extracting with chloroform and extracting with chloroform:isoamyl alcohol.

29. A method for isolating RNA from a biological sample comprising: mixing a biological sample with an extraction reagent to form a homogenate, wherein the biological sample and the extraction reagent are combined at a ratio of less than 50 mg of the biological sample to 1 ml extraction reagent, and wherein the extraction reagent comprises a reducing agent; extracting the RNA with an organic solvent; and precipitating RNA from the aqueous phase.

30. The method of claim 29 wherein the biological sample is an RNase-rich tissue.

31. The method of claim 30 wherein the RNase-rich tissue is pancreas or spleen.

32. The method of claim 29 wherein the extraction reagent comprises a guanidinium compound and phenol.

33. The method of claim 32 wherein the guanidinium compound is guanidine isothiocyanate.

34. The method of claim 29 wherein the extraction reagent is sold under the tradename Trizol® or Tri Reagent®.

35. The method of claim 29 wherein the precipitating comprises adding an alcohol and recovering the RNA.

36. The method of claim 29 wherein no greater than 25 mg of the biological sample is mixed with 1 ml extraction reagent.

37. The method of claim 29 wherein the reducing agent is β- mercaptoethanol.

38. The method of claim 37 wherein β-mercaptoethanol is present in the extraction reagent at 1 % (vol/vol).

39 The method of claim 29 wherein the organic solvent comprises phenol and chloroform in a ratio of between 4 parts phenol to 1 part chloroform (4: 1) and 6 parts phenol to 1 part chloroform (6:1)

40. The method of claim 39 wherein the phenol has a pH between 4.1 and

4.5.

41. The method of claim 29 wherein the extracting comprises chloroform as the organic solvent.

42. The method of claim 29 wherein the extracting comprises chloroform and isoamyl alcohol as the organic solvent.

43. The process of claim 39 wherein the extracting further comprises extracting with chloroform and extracting with chloroform and isoamyl alcohol.

44. A kit for isolating RNA from a biological sample comprising in separate containers: an extraction reagent, an organic solvent, and an RNase-free buffer.