JP2008086283A - RNA extraction method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for extracting an RNA without containing a DNA derived from a cell with high accuracy upon extraction of the RNA from the cell containing the DNA. <P>SOLUTION: Disclosed is a method for extracting an RNA from a cell containing the DNA, wherein a nucleic acid-containing material containing the DNA is added in an extraction process of the RNA. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for extracting RNA from cells or the like.

  In recent years, the elucidation of functions at the molecular level of living organisms has progressed due to advances in various analysis techniques. In particular, the disease-related phenomena are being actively addressed because of the great effects of their clarification. There are a variety of substances that control organisms, such as nucleic acids, proteins, sugars, and hormones. Among them, nucleic acids such as DNA and mRNA have been studied, and their relation to various diseases has been elucidated.

  For example, various studies have been conducted on “cancer”, one of the important diseases, and mRNA is expressed using the fact that the expression level of mRNA of a specific gene differs between cancer cells and normal cells. A technique for detecting a minute amount of cancer cells by quantification has been devised.

  In addition, nucleic acid analysis methods have made remarkable progress in recent years, and even a very small amount of mRNA can be strictly analyzed, as represented by GeneChip of Affymetrix. As a result, it is possible to know the presence and amount of a very small amount of mRNA, as well as the existence of genes that have not been known so far.

  What is important in performing mRNA analysis is the extraction of mRNA from the provided specimen. In many cases, the specimen is a tissue piece or a cell suspension, but the mRNA component in the specimen is extracted by centrifugation, homogenization or the like. In any case, since the specimen is often provided in the state of a cell having a wall, in order to extract RNA components in the cell, the cell wall is crushed and the RNA or mRNA contained in the cell wall Need to be extracted.

  By the way, RNA is rapidly degraded when exposed to RNase. Since RNA-degrading enzymes are present in a relatively large number of places even in an experimental environment, attention is required for RNA extraction work. RNase-free or inactivated extraction kits or dedicated extraction reagents are supplied from various manufacturers, and RNA extraction and purification are generally performed using them. By using these kits, a very small amount of mRNA can be recovered with a high yield without reducing the yield.

  Many RNA recovery methods are known. The AGPC method, silica membrane method, anion exchange method, affinity purification method and the like are generally well known. The AGPC method is described in detail, for example, in Non-Patent Document 1, and the outline thereof is as follows. (1) A solution containing guanidine thiocyanate, phenol and chloroform is added to a biological material such as a cell to destroy a cell membrane and a cell wall, denature the contained protein, and extract DNA into an organic solvent phase. (2) Only the aqueous phase containing RNA is recovered by centrifugation. (3) RNA is precipitated by adding ethanol or isopropanol to the aqueous phase (ethanol precipitation or isopropanol precipitation). (4) Further, only RNA is separated by centrifugation.

Details of the technology regarding the silica membrane method are described in, for example, Patent Document 1. Nucleic acids can be extracted from biological materials such as cells in one step, and a low concentration buffer such as water or TE buffer is used as an eluent. Therefore, the extracted nucleic acid can be used directly for subsequent analysis without passing through desalting and concentration operations such as ethanol precipitation.
JP-A-2-289596 Analytical Biochemistry 162, 156-159 (1987)

  As already mentioned, the progress of nucleic acid analysis technology including expression analysis has been remarkable, and for example, much has been found in elucidating the mechanism of cancer cells. In particular, it has been found that even genes that have not attracted attention because of their low abundance so far are important genes for cancer characterization and differentiation from normal cells.

  Usually, in order to measure mRNA, an enzyme reverse transcription reaction or the like is performed, a single-stranded DNA is synthesized, and then its sequence and content are measured using molecular biology techniques such as quantitative PCR. Many. The reason is that RNA is rapidly degraded by RNase and that reverse transcription is better for PCR or cRNA synthesis.

  When a very small amount of mRNA is to be measured, contamination of DNA derived from cells must be avoided. This is because mRNA is synthesized by transcription from DNA, and at least one copy of each partial sequence of mRNA exists in DNA.

  The RNA extraction method described in the background art has also been devised to prevent DNA contamination. In the AGPC method, the liquid volume ratio between the organic phase and the aqueous phase and the pH value are strictly controlled. The silica membrane method is also devised such as repeated column washing. However, even if these methods are used, it is difficult to completely remove DNA having similar physical properties. In order to remove the contaminated DNA, treatment with a DNA degrading enzyme (DNase) or the like may be added, but the operation is complicated and the recovery rate of RNA may be reduced. Further, since the degree of purification of the DNA degrading enzyme itself is low and RNA degrading enzymes may be contained, it is not particularly suitable for extracting a very small amount of mRNA.

  Therefore, development of a highly accurate RNA extraction method that does not contain cell-derived DNA has been desired.

  As a result of repeated studies to solve the above problems, it has been found that in the process of extraction from cells, it is possible to significantly reduce contamination of cell-derived DNA components by adding a DNA-containing material not derived from cells.

  In the step of purifying (extracting) RNA from a mixture of RNA and DNA, the conditions are set such that only RNA remains in the solution or on the surface of the carrier, utilizing the difference in physical properties of DNA and RNA. However, even under conditions that exclude DNA (DNA does not exist), DNA cannot be completely eliminated, and dissolution or adsorption of a certain amount of DNA is unavoidable even in a very small amount.

  Therefore, the present invention is intended to increase the absolute amount of total DNA by adding harmless DNA, relatively reduce the proportion of cell-derived DNA, and reduce the absolute amount of cell-derived DNA in a “constant amount”. It is. That is, the mechanism of the present invention is to replace DNA that is slightly mixed with harmless DNA (DNA that is not derived from cells) with completely different DNA that is not derived from cells.

  Therefore, the RNA extraction method according to the present invention is a method for extracting RNA from a mixture containing cell-derived DNA and other contaminants. At the time of purification of RNA to be extracted, a nucleic acid mixture containing DNA as a main component is extracted. It is an extraction method of RNA characterized by adding.

  The RNA extraction method of the present invention is a method for extracting RNA from a mixture containing cell-derived DNA and other contaminants, and a substrate or primer DNA used in a reverse transcription reaction performed after RNA extraction is added in the RNA extraction step. This is a method for extracting RNA.

  According to the present invention, it is possible to provide an RNA extraction method capable of reducing the contamination of cell-derived DNA components to zero at the time of RNA extraction from tissue pieces or cells. The method for purifying RNA according to the present invention can prevent contamination of DNA contained in a sample to be measured, and therefore, a very small amount of mRNA that is greatly contaminated with a small amount of DNA and that impairs quantification can be measured. In the detection and analysis of cancer cells, etc., detection and analysis (diagnosis) may be performed depending on the presence or absence of a small amount of mRNA, but it is high quality by accurately detecting a small amount of mRNA contained in the cell. Diagnosis and analysis are possible.

  As a biological sample from which RNA can be extracted using the present invention, any biological sample containing RNA can be used. That is, tissue sections collected from animals including humans, cell suspensions in which cells float, pellets obtained by aggregating them, artificially cultured cells, etc. can be mentioned, and these may be in either liquid or solid form . It can also be applied to RNA extraction from biological samples other than animal tissues such as bacteria, plants and viruses. Moreover, even if impurities other than the biological sample to be extracted with RNA are mixed, the RNA extraction method of the present invention can be applied without any obstacles. For example, whole blood, serum, saliva, urine, semen and the like including leukocytes and erythrocytes are also applicable.

  RNA extraction from cells or the like usually involves contamination of DNA, protein, lipid, etc., and various RNA extraction methods are employed. Typical techniques include AGPC method in which DNA is extracted into an organic solvent by controlling pH, and silica membrane method in which RNA is adsorbed on a carrier such as silica gel to remove unnecessary components including cell-derived DNA.

  In the AGPC method, a solution of guanidine thiocyanate is added to crush cell walls or the like and inactivate RNase, and then 2M sodium acetate, water-saturated equilibrium acidic phenol and chloroform are added and centrifuged. As a result, RNA is extracted into the liquid aqueous phase separated into two phases except for the intermediate phase, and DNA is extracted into the organic phase.

  The DNA-containing material of the present invention is preferably added in the form of an aqueous solution, and can be added at any stage of the RNA extraction process. The greater the efficiency of replacing cell-derived DNA contained in the aqueous phase, the greater the effect of removing cell-derived DNA according to the present invention. Therefore, it is preferable to add DNA at the earliest possible step and add a solution of guanidine thiocyanate. It is preferred to add it directly to the sample before.

  The amount and concentration of the DNA solution to be added are not particularly limited, but when an extraction reagent or the like is prepared in advance, it is desirable to adjust the amount of the solution within a range that does not affect the extraction accuracy. In general, the extraction reagent is optimized so that RNA can be extracted with high purity. Therefore, the effect of reducing the amount of the nucleic acid-containing solution to be added, that is, the amount of water brought in is as small as possible.

  As the DNA to be added, any DNA can be used as long as the sequence has little influence on the use (measurement, quantification, analysis) of the extracted RNA. It may be a synthesized DNA, or a natural or non-natural DNA. For example, Poly-A, which is a polymer of adenine, and Poly-T, which is a polymer of thymine, can be used as the nucleic acid used in the present invention. It is also possible to use a biological species different from the RNA biological species to be extracted. In that case, any nucleic acid-containing material that does not contain hindered DNA depending on the use of the extracted RNA is used. be able to.

When extracting mRNA for the purpose of expression analysis, etc., cDNA is synthesized from the extracted RNA by a reverse transcription reaction, or cDNA having a promoter sequence is synthesized and cRNA is synthesized using the cDNA as a template. Sometimes. In such a case, DNA necessary for the reaction is used in the reverse transcription reaction step, but the DNA used therein can be used as a part or all of the nucleic acid-containing material of the present invention. For example, in the Superscript series reverse transcriptase commercially available from Invitrogen, it is recommended to use 12 to 18-mer oligo dT or 5 to 9-mer random primer as a reverse transcription primer. In this case, those DNAs can be used as added DNAs during RNA extraction of the present invention. In addition, when cRNA synthesis is performed, a promoter sequence is required for the cDNA to be synthesized, and therefore a primer having a promoter sequence is often used during reverse transcription reaction. Various types of promoters are known, and main promoters include SP6, T7, T3, and the like. Among these, the T7 promoter is widely used, and the following primers are used for reverse transcription as primers for this purpose.
5'GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT3 '(SEQ ID NO: 1)

  In the RNA extraction according to the present invention, the DNA of SEQ ID NO: 1 can be used.

  In addition, when reverse transcription is performed using gene-specific primers, it can also be used as DNA to which those primers are added.

  When a reverse transcription reaction is performed after RNA extraction, and when directly labeling with a fluorescent dye, a radioisotope, or the like, it is possible to label the added DNA.

  The AGPC method and extraction based on the same principle are widely used, and many related reagents and kits are commercially available. ISOGEN, commercially available from Nippon Gene, is a widely used kit. Even when the kit is used, it is possible to apply the RNA extraction method of the present invention. When adding ISOGEN to cells or tissue fragments, the DNA solution of the present invention should be added in advance. Is preferred. In this case, it is preferable to add the DNA in as small a volume as possible so as not to significantly change the liquid composition of ISOGEN.

  In addition, when extracting RNA with a higher degree of purification, RNA extraction may be performed a plurality of times using the RNA obtained in the first extraction as a sample. It can also be applied in situations.

  The RNA extraction according to the present invention can also be applied to RNA extraction other than the AGPC method. As for the silica membrane method, kits using the method are commercially available from Qiagen, Agilent, and the like. The silica membrane method is an RNA extraction method using a selective adsorption mechanism to a silica gel membrane under control of ion conditions. Strictly speaking, since DNA and RNA are selected based on the difference in salt concentration, contamination derived from cell-derived DNA into the extracted RNA is unavoidable. The RNA extraction method according to the present invention can prevent contamination of genome-derived DNA by adding non-intrusive DNA to contaminants containing cell-derived genome.

  When the RNA extraction method of the present invention is used in the silica membrane method, it is also effective to add DNA in advance to the silica membrane before the sample is applied. That is, by applying DNA to a silica membrane in advance, it can be saturated with DNA to which physical adsorption or binding has been added.

  In addition to the AGPC method and the silica membrane method, the RNA extraction method of the present invention can be used for anion exchange method, affinity purification method, and the like. In any extraction method, it is possible to significantly reduce contamination of cell-derived DNA by adding DNA applicable in the present invention to a target specimen. Similarly to the silica membrane method, when a carrier for extraction and purification is present, it is more effective to apply the DNA to the carrier in advance. Mixing can be avoided.

  In RNA extraction methods other than the AGPC method such as silica membrane method, anion exchange method, and affinity purification method, various types of DNA can be selected as the type of DNA to be added, as in the AGPC method. Moreover, labeled DNA, a primer for reverse transcription reaction, etc. can be used suitably.

  Hereinafter, the present invention will be described in more detail with reference to specific examples.

<Example 1>
(1) Total RNA recovery from cells and cDNA preparation (1-1) Total RNA recovery using ISOGEN (AGPC method) Gastric cancer-derived cell line (cell line) KATO III collected from human stomach In accordance with the above, culture was performed to obtain about 1 × 10 ⁵ cultured cells. The obtained cultured cells were centrifuged, pelletized, and the cells were collected by removing the upper scum.

Subsequently, DNA having the following base sequence was synthesized. After synthesizing according to a usual method and purifying by HPLC, it was dissolved in water so that the final concentration was 100 μM.
5'GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT3 '(SEQ ID NO: 1)
50 μl of the obtained DNA solution was added to the cells collected in a pellet form.

  Subsequently, using an RNA extraction kit “ISOGEN” manufactured by Nippon Gene, 500 μl of ISOGEN was added to suspend the pellet. The suspension was sufficiently suspended using a stirrer or the like and then allowed to stand at room temperature for 5 minutes.

  Next, 100 μl of chloroform was added and stirred well again, and then allowed to stand at room temperature for several minutes.

  Using a micro high-speed centrifuge set at 4 ° C., the mixture was centrifuged at 12,000 g for 15 minutes, and the aqueous phase component accumulated at the top was collected. The recovery was performed with care so as not to remove an intermediate portion between the phenol phase and the aqueous phase.

  The recovered aqueous phase component was transferred to another tube, and 500 μl of isopropanol was added to the tube. The pellet was lightly rinsed with isopropanol, allowed to stand at room temperature for about 10 minutes, and centrifuged at 12000 g for 10 minutes using a micro high speed centrifuge set at 4 ° C.

  Care was taken not to suck the pellet components that had settled at the bottom of the tube, the top isopropanol was removed by suction, and 500 μl of ethanol adjusted to a concentration of 70% was added. As with isopropanol, the pellet was lightly rinsed with an ethanol solution, and centrifuged at 7500 g for 5 minutes using a micro high-speed centrifuge set at 4 ° C. Care was taken not to aspirate the pellet components that had settled to the bottom of the tube, the ethanol at the top was removed and air dried briefly.

(1-2) Synthesis of cDNA 0.2 μg of the total RNA obtained was reversed by a primer having the T7 promoter sequence (same as the nucleic acid of SEQ ID NO: 1) using SuperScript Choice System for cDNA Synthesis manufactured by Invitrogen. I did a copy. Specifically, the following method was used. The reaction was prepared so that the final liquid volume was 20 μl.

  1 μl of a primer (100 μM) having a T7 promoter sequence was added to 0.2 μg of total RNA in a volume of 11 μl, and heat denatured at 70 ° C. for 10 minutes. After quenching, 4 μl of 5 × buffer attached to the kit, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP Mix were added, and then 1 μl of reverse transcriptase Superscript II RT was added and incubated at 42 ° C. for 1 hour. After 1 hour, it was cooled rapidly and about 20 μl of the 1st strand solution was recovered.

(1-3) Reverse transcription (synthesis of 2nd strand)
A total amount of about 20 μl of the obtained 1st strand solution was synthesized without any purification according to the manual of SuperScript Choice System for cDNA Synthesis. Specifically, 5 × buffer 30 μl, 10 mM dNTP Mix 3 μl, DNA ligase 1 μl (10 units), DNA polymerase I 4
After adding μl (40 units) and RNaseH 1 μl (2 units), the mixture was reacted at 16 ° C. for 2 hours. Since the double-stranded cDNA thus obtained was not blunt-ended, it was blunted with 2 μl (10 units) of T4 DNA polymerase.

  The resulting double-stranded cDNA was extracted and purified using phenol / chloroform / isoamyl alcohol, and the nucleic acid component was purified using a phase-locked gel. The obtained cDNA was dissolved in an appropriate amount of water, and the purity and yield were measured by absorbance measurement.

(2) Expression level quantification by quantitative PCR The expression level of the gene contained by quantitative PCR was measured using the obtained double-stranded cDNA. The measurement targets were CEA and CCL5 genes.

  The 7500 RealTime PCR System provided by Applied Biosystems was used as the quantitative PCR system.

  In order to quantify each gene, TaqMan Gene Expression Assay was used. As a probe for measurement, a prepared probe provided by Applied was used. Specifically, Hs00237075 was used for CEA measurement and Hs00174575 was used for CCL5.

PCR was performed using a 20 ng template, and the volume was 50 μl. TaqMan 2 × Universal Master Mix NO AmpErase UNG was used as the Master Mix for PCR.
The PCR cycle was a two-stage PCR cycle shown in Table 1 below.

Analysis is performed with the analysis software attached to the above quantitative PCR system, and the number of cycles when the value of Delta Rn, which is a numerical value on the software that increases according to the amount of PCR product, exceeds 0.1 is defined as the Ct value. Then, each gene was quantified.
The Ct values for CEA and CCL5 are shown in Table 2 below.

  From this result, it was found that KATOIII used as a sample in this example showed constant expression of CEA and no expression of CCL5 (lower than measurement sensitivity). From various reported databases, it is known that the expression of CCL is almost zero in KATOIII. Therefore, from this result, it was shown that there was no noise derived from DNA that cells had, not RNA, that is, there was no DNA contamination.

<Comparative Example 1>
The same experiment as in Example 1 was performed without adding the T7 primer. That is, RNA extraction was performed according to the usual treatment method without adding the T7 primer before the ISOGEN treatment.

  Except for not adding the T7 primer, extraction of total RNA and synthesis of double-stranded cDNA were carried out in the same manner as in Example 1. Quantitative PCR was also performed by the same system of Applied Biosystems. The measured Ct values are shown in Table 3 below.

  From this result, the RNA extract not subjected to the T7 primer treatment showed the expression of CCL5. As already mentioned, it is known that the expression of CCL is almost zero in KATOIII. Therefore, the expression of CCL5 shown in Comparative Example 1 is considered to be highly likely to be a DNA-derived signal.

Claims (11)

A method of extracting RNA from cells containing DNA,
In the RNA extraction process, a nucleic acid-containing material containing DNA is added.   The method for extracting RNA according to claim 1, wherein the nucleic acid-containing material contains DNA that is a substrate for a reverse transcription reaction performed after RNA extraction.   The method for extracting RNA according to claim 2, wherein the substrate DNA is a primer for a reverse transcription reaction performed after RNA extraction.   The RNA extraction method according to claim 3, wherein the primer for the reverse transcription reaction is a random primer having a chain length of 5 to 9. The RNA extraction method according to claim 3, wherein the reverse transcription reaction primer includes a primer having the following sequence.
5'GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT3 '(SEQ ID NO: 1)   The RNA extraction method according to claim 3, wherein the reverse transcription reaction primer comprises a gene-specific primer.   The RNA extraction method according to any one of claims 1 to 6, wherein the DNA to be added is labeled with a radioisotope or a fluorescent substance.   The RNA extraction method according to any one of claims 1 to 7, wherein the RNA extraction method is an AGPC method.   The RNA extraction method according to any one of claims 1 to 7, wherein the RNA extraction method is a silica membrane method.   The RNA extraction method according to any one of claims 1 to 7, wherein the RNA extraction method is an anion exchange method.   The RNA extraction method according to any one of claims 1 to 7, wherein the RNA extraction method is an affinity purification method.