JP4073447B2 - RNA extraction method, RNA extraction reagent, and biomaterial analysis method - Google Patents

Description

  The present invention relates to RNA extraction from RNA-containing biological materials and methods for analyzing RNA-containing biological materials.

  DNA is a substance responsible for all genetic information of organisms, while RNA is a substance that plays an important role in protein synthesis in vivo based on DNA genetic information. In recent years, DNA analysis has revealed gene sequence information of many species. Following this, the importance of elucidating gene functions by analyzing RNA has increased, and the operation of isolating RNA from biological materials has become indispensable. RNA analysis methods mainly include reverse transcription polymerase chain reaction (RT-PCR), Northern blot, and the like.

  In order to obtain good analysis results in these analysis methods, it is required to use high-purity RNA. Particularly in RT-PCR, RNA is difficult to analyze when RNA is mixed with DNA. Therefore, in RNA extraction from biological materials, it is desired that RNA is isolated with high purity from DNA, proteins, lipids, sugars and the like mixed in cells.

  There is an AGPC method as a general RNA extraction method. In the AGPC method, (1) a biological material is dissolved in a guanidine thiocyanate solution, an acidic buffer solution, a phenol solution, and a chloroform solution are sequentially added and mixed, and (2) an aqueous phase containing RNA, a denatured protein, and The organic solvent phase containing insolubilized DNA is separated into an intermediate layer between the aqueous phase and (3) ethanol or isopropanol is added to the aqueous phase containing RNA, and (4) the insolubilized RNA is selectively precipitated by centrifugation.

  Utilizes the property of nucleic acid binding to silica in the presence of a chaotropic agent as a nucleic acid extraction method that does not require a relatively long operation such as ethanol precipitation or isopropanol precipitation without using toxic poisons such as phenol and chloroform Thus, there are a method for recovering nucleic acid from an agarose gel and a method for extracting nucleic acid from a biological material using a chaotropic agent and silica particles. However, these methods do not have RNA and DNA selectivity, and the extracted nucleic acid is a mixture of RNA and DNA. Therefore, an operation for removing DNA contained in the extracted nucleic acid may be necessary for RNA analysis. DNA removal is mainly performed by a DNA-degrading enzyme treatment, and then an enzyme removal operation is performed as necessary. The DNA removal operation generally requires a processing time of about 1 hour due to a DNA degrading enzyme. In addition, enzyme removal mainly requires complicated operations such as phenol / chloroform extraction and ethanol precipitation, resulting in RNA loss.

  There is a method for selectively extracting RNA using the property that RNA binds to silica in the presence of a chaotropic agent and an organic solvent (Patent Document 1 below). In this method, a polar organic solvent such as ethanol or isopropanol is added to a chaotropic agent to control the difference in binding characteristics between DNA / RNA and silica, and RNA is selectively bound to silica. However, even in this method, RNA selectivity is insufficient, and an operation for removing DNA mixed in the extracted nucleic acid is required.

JP 2002-187897 A

  An object of the present invention is to provide a method for selectively extracting and analyzing high-purity RNA from biological materials containing RNA by a safe, rapid and simple operation.

  The inventors found that RNA binds to silica with extremely high selectivity in the presence of a predetermined concentration of a chaotropic agent and a predetermined concentration of an organic solvent, and established the RNA selective extraction / analysis method of the present invention. It came.

  In the present invention, a biological material containing RNA in a free state is mixed with a predetermined concentration of a chaotropic agent and a predetermined concentration of an organic solvent, and the mixed solution is brought into contact with a nucleic acid-binding solid phase so that the nucleic acid-binding solid phase to which RNA is bound. It relates to washing the phase and eluting the RNA from the nucleic acid binding solid phase to which the RNA is bound. The present invention also relates to analyzing the obtained RNA by reverse transcription polymerase chain reaction (RT-PCR).

  According to the present invention, RNA can be extracted with extremely high purity. In addition, since the extract contains almost no DNA, RT-PCR, which is a DNA-sensitive RNA analysis method, can be performed without performing DNA removal operations that may damage RNA, and biological samples can be obtained with high accuracy. Can be analyzed.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings. It should be noted that the drawings are used exclusively for explanation and do not limit the scope of rights.

  The biological material containing RNA may be a biological sample such as whole blood, serum, sputum, urine, biological tissue, cultured cells, cultured bacteria, or a substance containing RNA in a purified state. it can.

  The biological material can be dissolved by a physical method using a mortar, ultrasonic wave, microwave, homogenizer, etc., a chemical method using a surfactant or a protein denaturant, or a biochemical method using a proteolytic enzyme, And it is performed by the method which combined them.

  Preferred examples of the chaotropic agent include sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, and guanidine hydrochloride.

  In addition, as the organic solvent, one or a combination of two or more compounds having 2 to 10 carbon atoms selected from aliphatic ethers, aliphatic esters, and aliphatic ketones can be used.

  As aliphatic ethers, preferably ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propion glycol dimethyl ether, propion glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran and 1.4-dioxane are used.

  As the aliphatic ester, propylene glycol monomethyl ether acetate or ethyl lactate is preferably used.

  As the aliphatic ketone, preferably acetone, hydroxyacetone, or methyl ketone is used.

  The RNA selective extraction method of the present invention is based on the selective binding effect between silica and RNA, and this effect is obtained in the presence of a predetermined concentration of a chaotropic agent and a predetermined concentration of an organic solvent.

  When guanidine thiocyanate is used as the chaotropic agent and diethylene glycol dimethyl ether is used as the organic solvent, high-purity RNA can be recovered at a good recovery rate when the guanidine thiocyanate concentration is 1.0 to 4.0 mol / l and the diethylene glycol dimethyl ether concentration is 10 to 30%. can get. In particular, when the guanidine thiocyanate concentration in the mixed solution is 1.5 to 2.0 mol / l and the diethylene glycol dimethyl ether concentration is 15 to 25%, extremely high-purity RNA can be obtained with a high recovery rate.

  In addition, when guanidine thiocyanate is used as the chaotropic agent and ethyl lactate is used as the organic solvent, high purity RNA is recovered at a guanidine thiocyanate concentration of 1.0 to 4.0 mol / l and an ethyl lactate concentration of 20 to 40% in the mixed solution. It is obtained by. In particular, extremely high-purity RNA can be obtained with a high recovery rate at a guanidine thiocyanate concentration of 1.5 to 2.5 mol / l and an ethyl lactate concentration of 25 to 35% in the mixed solution.

  Examples of the nucleic acid-binding solid phase preferably include substances containing silicon oxide such as glass particles, silica particles, glass fiber filter paper, silica wool, crushed materials thereof, and diatomaceous earth.

  The contact between the mixed solution and the nucleic acid-binding solid phase is performed by a method of stirring and mixing the solid phase and the mixed solution in a container, or a method of passing the mixed solution through a column or the like on which the solid phase is immobilized. After bringing the solid phase into contact with the mixed solution, the solid phase and the mixed solution are separated.

  The washing of the nucleic acid-binding solid phase to which the nucleic acid has been bound is performed, for example, by bringing the washing solution into contact with the solid phase and then separating the washing solution from the solid phase. As the washing solution, it is preferable to use ethanol having a concentration of at least 75% so as not to elute the nucleic acid bound to the solid phase and to efficiently remove non-specifically bound substances.

  The means for eluting the nucleic acid from the nucleic acid-binding solid phase is carried out by contacting the eluent with the solid phase, eluting the nucleic acid bound to the solid phase into the eluent, and then separating the eluent from the solid phase. As an eluent, water or a low salt concentration buffer solution that has been subjected to RNase removal or RNase inactivation treatment is used. Moreover, elution efficiency improves by performing elution under heating.

  The eluate from which the nucleic acid has been eluted can also be used immediately for reverse transcription polymerase chain reaction.

  In this example, RNA is extracted from cultured cells using guanidine thiocyanate as a chaotropic agent and diethylene glycol dimethyl ether as an organic solvent.

<Extraction of RNA>
As a first step, the pellets of the mouse Myeloma (manufactured by Dainippon Pharmaceutical Sp / 0-Ag14) cultured cells (10 ⁶ or so), cell lysates (guanidine thiocyanate 4mol / l, 10mmol / l MES -KOH, pH6 .5) 600 μl was added, and the cells were disrupted with a homogenizer (HANDY MICRO HOMOGENIZER manufactured by Microtech Nichion Co., Ltd.) to release the nucleic acid in the cells.

  As the second step, 600 μl of diethylene glycol dimethyl ether aqueous solution (20, 40, 60, 80, 100 vol%) is added as a polar organic solvent to the cell lysate containing the nucleic acid in the free state. At this time, the concentration of guanidine thiocyanate in the mixed solution is 2 mol / l, and the concentration of diethylene glycol dimethyl ether is 10, 20, 30, 40, 50% by volume.

  As a third step, as shown in FIG. 1, a syringe (25 ml syringe manufactured by Terumo Corporation) is used in a chip for nucleic acid capture in which 5 mg of silica wool (B grade manufactured by Toshiba Chemical Co., Ltd.) is filled as the nucleic acid-binding solid phase at the tip of the polypropylene chip. ), The nucleic acid and the solid phase are brought into contact with each other and separated by aspirating and discharging a solution containing nucleic acid in a free state, cell lysate, and diethylene glycol dimethyl ether.

  As a fourth step, 1200 μl of the washing solution (80% by volume ethanol aqueous solution) is aspirated and discharged by the nucleic acid capturing chip, so that the solid phase and the washing solution are brought into contact with each other and separated to remove non-specifically bound substances on the solid phase. .

  As a fifth step, 100 μl of the eluent (DEPC-treated water) is aspirated and discharged by the nucleic acid capturing chip, whereby the solid phase and the eluent are brought into contact with each other and separated to obtain an eluent containing the purified nucleic acid.

<Evaluation of extracted RNA>
A portion of the eluate was electrophoresed on a 1.25% agarose gel (Reliant RNA Gel System, manufactured by FMC), and after ethidium bromide staining, a photograph was taken under UV irradiation using a transilluminator. . Lanes 1 and 2 use 40% by volume diethylene glycol dimethyl ether aqueous solution, lanes 3 and 4 use 60% by volume diethylene glycol dimethyl ether aqueous solution, lanes 5 and 6 use 80% by volume diethylene glycol dimethyl ether aqueous solution, lane 7 , 8 shows an extracted nucleic acid when 100% by volume of diethylene glycol dimethyl ether aqueous solution is used.

  Nucleic acids are separated according to molecular weight by electrophoresis, and genomic DNA, 28SrRNA, 18SrRNA, and tRNA bands are shown from the top. FIG. 2 shows that when 40% by volume of diethylene glycol dimethyl ether aqueous solution is used, almost no genomic DNA is observed and RNA with extremely high purity can be obtained with a high recovery rate. On the other hand, when 100 to 100 volume% of diethylene glycol dimethyl ether aqueous solution 60 is used, it is indicated that the extracted nucleic acid contains a large amount of genomic DNA. When 20% by volume of diethylene glycol dimethyl ether aqueous solution was used, almost no extracted nucleic acid was obtained.

  In this example, RNA is extracted from cultured cells using guanidine thiocyanate as a chaotropic agent and ethyl lactate as an organic solvent.

<Extraction of RNA>
The same process as in Example 1 was performed except for the second step. Hereinafter, the second step will be described.
As a 2nd process, 600 microliters of ethyl lactate aqueous solution (20, 40, 60, 80, 100 volume%) is added as a polar organic solvent to the cell lysis solution containing a nucleic acid of a free state. At this time, the concentration of guanidine thiocyanate in the mixed solution is 2 mol / l, and the concentration of ethyl lactate is 10, 20, 30, 40, 50% by volume.

<Evaluation of extracted RNA>
The result of electrophoresis performed in the same manner as in Example 1 is shown in FIG. Lane 1 shows the extracted nucleic acid when 60% by volume of ethyl lactate aqueous solution was used, Lane 2 shows the extracted nucleic acid solution when 80% by volume of ethyl lactate aqueous solution was used, and Lane 3 shows the extracted nucleic acid when 100% by volume of ethyl lactate aqueous solution was used.

  This shows that when 60% by volume of an ethyl lactate aqueous solution is used, almost no genomic DNA is observed and RNA with extremely high purity can be obtained with a high recovery rate. On the other hand, when 80% and 100% by volume of ethyl lactate aqueous solution is used, it is shown that the extracted nucleic acid contains a large amount of genomic DNA. In addition, when using 20 and 40% by volume of ethyl lactate aqueous solution, almost no extracted nucleic acid was obtained.

Comparative example

  RNA was extracted from cultured cells using an RNA extraction kit (RNeasy Mini Kit manufactured by QIAGEN) using guanidine thiocyanate as a chaotropic agent and ethanol as an organic solvent. This method is based on the method described in Patent Document 1.

<Extraction of RNA>
From the pellet of the same mouse Myelom cultured cells of Example 1 ⁽¹⁰⁶ or so), using the QIAGEN Inc. RNeasy Mini Kit, according to the protocol attached to the kit, RNA extraction was performed.

<Evaluation of extracted RNA>
The result of electrophoresis performed in the same manner as in Example 1 is shown in FIG. From this, it was shown that genomic DNA was contained in the extracted nucleic acid when the RNeasy Mini Kit was used.

<RT-PCR using extracted nucleic acid>
RT-PCR was performed using the extracted nucleic acid obtained in Example 1 and the extracted nucleic acid obtained by the method of the Comparative Example.
A nucleic acid solution containing 2.5 μg of total RNA was prepared from the extracted nucleic acids obtained by the methods of Example 1 and Comparative Example without performing the DNA removal operation. To this nucleic acid solution, reverse transcriptase (Invitrogen SuperScriptII) and reverse transcription reagent containing Oligo (dT) primer are added to make a final solution volume of 20 μl and incubated at 42 ° C. for 50 minutes. CDNA as a template was synthesized.

  Next, PCR primers (mouse β-actin RT-PCR Primer Set, manufactured by Toyobo Co., Ltd.), thermostable DNA polymerase (toyobo Co., Ltd.) targeting the mouse β-actin gene region containing no intron were added to 2 μl and 0.2 μl of the solution after reverse transcription reaction. (Applied Biosystems AmpliTaq Gold DNA polymerase) and PCR reagent are added to make the final volume 50 μl, and thermal cycler (94 ° C, 15 seconds, 55 ° C, 30 seconds, 72 ° C, 1 minute 30 cycles) PERAIN ELMAR GeneAmp PCR System 9600) was used.

  In addition, as a negative control, 2 μl and 0.2 μl of a reverse transcription unreacted solution that does not perform a reverse transcription reaction, and a mouse attached to a PCR primer (Toyobo Mouse β-actin RT-PCR Primer Set) as a positive control PCR was performed using β-actin-derived DNA.

  The solution after the PCR reaction was electrophoresed on a 3% agarose gel (FMC Nusieve 3: 1 Agarose), and after ethidium bromide staining, a photograph was taken under UV irradiation using a transilluminator. .

  In FIG. 5, lane 1 is an amplification product when a reverse transcription reaction is performed using the extracted nucleic acid obtained by the method of Example 1 and PCR is performed from 2 μl of the solution after the reverse transcription reaction, and lane 2 is a method of Example 1. A reverse transcription reaction is performed using the extracted nucleic acid obtained by the above step, and the amplified product in the case of PCR from 0.2 μl of the solution after the reverse transcription reaction, Lane 3 shows the reverse transcription reaction using the extracted nucleic acid obtained by the method of Example 1. Without amplification, amplification product when PCR was performed from 2 μl of the reverse transcription unreacted solution, lane 4 did not perform reverse transcription reaction using the extracted nucleic acid obtained by the method of Example 1, and PCR was performed from 0.2 μl of the reverse transcription reaction non-reactive solution. The amplification product is shown.

  Lane 5 is a reverse transcription reaction using the extracted nucleic acid obtained by the method of the comparative example, and the amplified product obtained by PCR from 2 μl of the solution after the reverse transcription reaction. Lane 6 is the nucleic acid obtained by the method of the comparative example. Amplified product when PCR was performed from 0.2 μl of the solution after reverse transcription reaction, Lane 7 is the reverse transcription reaction without using the extracted nucleic acid obtained by the method of the comparative example and performing the reverse transcription reaction. Amplification product when PCR is performed from 2 μl of the solution, Lane 8 shows the amplification product when PCR is performed from 0.2 μl of the reverse transcription unreacted solution without performing the reverse transcription reaction using the extracted nucleic acid obtained by the method of the comparative example. Lane 9 shows an amplification product when PCR is performed using mouse β-actin-derived DNA as a positive control.

  As a result, in lanes 1, 2, 5, 6, 7, and 9, a 540 bp amplification product derived from the mouse β-actin gene was confirmed. In the extracted nucleic acid obtained by the method of Example 1, the amplification product (lanes 3 and 4) was not confirmed when the reverse transcription reaction was not performed. Therefore, the amplification product (lanes) when the reverse transcription reaction was performed. 1 and 2) are derived from mRNA, indicating that RT-PCR is possible without removing genomic DNA from the extracted nucleic acid.

  On the other hand, in the extracted nucleic acid obtained by the method of Comparative Example, an amplification product (lane 7) was obtained when 2 μl of the reverse transcription unreacted solution that did not perform the reverse transcription reaction was used. This is an amplification product derived from genomic DNA contained in the extracted nucleic acid, and the PCR amplification product (lane 5) using 2 μl of the reverse transcription reaction solution subjected to reverse transcription was amplified from mRNA and genomic DNA. It becomes a mixture of products, indicating that normal RT-PCR cannot be performed. Therefore, in order to perform RT-PCR from the extracted nucleic acid obtained by the method of the comparative example, it is necessary to remove the genomic DNA of the extracted nucleic acid.

The nucleic acid capturing chip used in Example 1 and Example 2. The electrophoresis result of the extracted nucleic acid in Example 1. The electrophoresis result of the extracted nucleic acid in Example 2. The electrophoresis result of the extracted nucleic acid in a comparative example. Electrophoresis result of RT-PCR product.

Claims (16)

A nucleic acid-binding solid phase containing ethyl lactate, a chaotropic agent, and silicon oxide is mixed with a biological material containing RNA to selectively bind RNA to the solid phase,
Separating the solid phase bound to RNA from the liquid phase;
Washing the solid phase,
An RNA extraction method for eluting RNA from a solid phase, comprising:
The concentration of the chaotropic agent in the mixed solution consisting of the biological material containing RNA, ethyl lactate and the chaotropic agent is 1.0 to 4.0 mo1 / 1,
How ethyl lactate concentration in the mixed solution is 35% 20.   The RNA extraction method according to claim 1, wherein the chaotropic agent is at least one selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, and guanidine hydrochloride. 2. The RNA extraction method according to claim 1, wherein the predetermined concentration of the guanidine thiocyanate is a concentration in a mixed liquid composed of a biological material containing RNA, ethyl lactate, and a chaotropic agent, and is 1.0 to 4.0 mol / a l, RNA extraction methods ethyl lactate concentration in the mixed solution is 35% 20.   2. The RNA extraction method according to claim 1, wherein the predetermined concentration of the guanidine thiocyanate is a concentration in a mixed solution composed of a biological material containing RNA, ethyl lactate, and a chaotropic agent. The RNA extraction method wherein the concentration of ethyl lactate in the mixed solution is 25 to 35%.   The RNA extraction method according to any one of claims 1 to 4, wherein the nucleic acid-binding solid phase containing silicon oxide is glass particles, silica particles, glass fiber filter paper, silica wool, or a crushed material thereof. RNA extraction method which is diatomaceous earth.   6. The RNA extraction method according to claim 1, wherein the biological material containing the RNA is whole blood, serum, sputum, urine, biological tissue, cultured cells, or cultured bacteria. .   The RNA extraction method according to any one of claims 1 to 6, wherein the washing liquid is ethanol.   The RNA extraction method according to any one of claims 1 to 7, wherein the eluent is water or low salt concentration buffer solution that has been subjected to RNase removal or RNase inactivation treatment. Method. A nucleic acid-binding solid phase containing ethyl lactate, a chaotropic agent, and silicon oxide is mixed with a biological material containing RNA to selectively bind RNA to the solid phase,
Separating the solid phase bound to RNA from the liquid phase;
Washing the solid phase,
Eluting RNA from the solid phase,
A biological material analysis method for amplifying the obtained RNA by reverse transcription polymerase chain reaction (RT-PCR),
The concentration of the chaotropic agent in the mixed solution consisting of the biological material containing RNA, ethyl lactate and the chaotropic agent is 1.0 to 4.0 mo1 / 1,
How ethyl lactate concentration in the mixed solution is 35% 20.   The method for analyzing a biological material according to claim 9, wherein the chaotropic agent is at least one selected from sodium iodide, potassium iodide, sodium thiocyanate, guanidine thiocyanate, and guanidine hydrochloride. . 10. The method for analyzing a biological material according to claim 9, wherein the predetermined concentration of guanidine thiocyanate is a concentration in a mixed solution composed of an RNA-containing biological material, ethyl lactate, and a chaotropic agent. 0 mol / a l, a method analyzing biological materials ethyl lactate concentration in the mixed solution is 35% 20.   10. The method for analyzing a biological material according to claim 9, wherein the predetermined concentration of guanidine thiocyanate is a concentration in a mixed solution composed of an RNA-containing biological material, ethyl lactate, and a chaotropic agent. The analysis method of the biological material which is 5 mol / l and whose ethyl lactate concentration in the said liquid mixture is 25 to 35%.   The method for analyzing a biological material according to any one of claims 9 to 12, wherein the nucleic acid-binding solid phase containing silicon oxide is glass particles, silica particles, glass fiber filter paper, silica wool, or their crushing. Of biological materials that are diatomaceous earth.   14. The biological material analysis method according to claim 9, wherein the biological material containing RNA is whole blood, serum, sputum, urine, biological tissue, cultured cells, or cultured bacteria. Material analysis method.   The biological material analysis method according to claim 9, wherein the cleaning liquid is ethanol.   16. The method for analyzing a biological material according to claim 9, wherein the eluent is water or a low salt concentration buffer solution that has been subjected to RNase removal or RNase inactivation treatment. Analysis method of biological materials.

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