JP2014039481A - Rna extraction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective method for extracting RNA.SOLUTION: The RNA extracting method comprises the steps of: mixing a solution containing a chaotropic substance and a solid carrier that binds nucleic acid with a sample containing ribonucleic acid (RNA) to cause adsorption of the RNA onto the carrier; washing the carrier adsorbing the RNA with a washing solution containing no organic solvent; and eluting the RNA adsorbed to the carrier into an elution solution containing a reverse transcriptase, dNTPs, and primer oligonucleotides.

Description

  The present invention relates to a method for extracting RNA.

Boom et al. Reported a method for more easily extracting nucleic acids from biomaterials using a nucleic acid-binding solid phase carrier such as silica particles and a chaotropic agent (see Non-Patent Document 1). Including the method of Boom et al., A method of adsorbing and extracting a nucleic acid on a carrier using a nucleic acid-binding solid phase carrier such as silica and a chaotropic agent mainly includes (1) a nucleic acid-binding solid phase in the presence of a chaotropic agent. A step of adsorbing nucleic acid to the phase carrier (adsorption step), (2) a step of washing the carrier adsorbed with nucleic acid with a washing solution (washing step) to remove non-specifically bound impurities and chaotropic agents, and ( 3) It consists of three steps of eluting nucleic acid from the carrier with water or a low salt concentration buffer (elution step). Here, as the washing liquid of (2), in order to dissolve the chaotropic agent and prevent elution of the nucleic acid from the carrier, conventionally, a water-soluble organic solvent, particularly ethanol, is used in an amount of 50 to 8.
Water or a low salt concentration buffer containing about 0% is used.

However, when this water-soluble organic solvent remains in the step (3), the enzyme reaction is inhibited when the extract is treated with an enzyme, and therefore, usually after washing with an aqueous solution containing ethanol, as necessary. An operation of washing with 100% ethanol or acetone with higher volatility and then drying to completely remove the organic solvent from the system is performed. Not only does this drying take time, but if the drying time is insufficient, ethanol will remain, and if it is excessive, the nucleic acid will be too dry and solidified, making it difficult to elute, resulting in nucleic acid. It is known that this leads to a decrease in the recovery amount and reproducibility. In this way, the use of organic solvents not only makes it difficult to determine the degree of drying, but organic solvents such as ethanol and acetone are flammable and volatile. There is also a danger.

Therefore, after the nucleic acid is adsorbed on the carrier, the carrier is washed with water or a low salt concentration buffer solution containing no organic solvent such as ethanol in the washing step (2), and 50% in the elution step (3). ~ 70
A method has been developed for extracting ribonucleic acid (RNA) by eluting the nucleic acid with water or a low salt concentration buffer at 0 ° C. (see Patent Document 1).

Japanese Patent Laid-Open No. 11-146783

J.Clin.Microbiol., Vol.28 No.3, p.495-503 (1990)

  An object of this invention is to provide the extraction method of RNA which can be utilized efficiently.

Until now, in the Boom method, the nucleic acid is adsorbed onto the carrier, and in the washing step (2), the carrier is washed with water or a low salt concentration buffer that does not substantially contain an organic solvent such as ethanol. In the elution step, the nucleic acid is eluted with water or a low salt concentration aqueous solution at 50 to 70 ° C.
Ribonucleic acid (RNA) has been isolated (Japanese Patent Laid-Open No. 11-146783). However, the present inventor has found that the subsequent reverse transcriptase reaction can be efficiently carried out by eluting the nucleic acid with a reverse transcriptase reaction solution or the like in the elution step (3), and the present invention has been completed.

In one embodiment of the present invention, a sample containing RNA is mixed with a lysate containing a chaotropic substance and a nucleic acid-binding solid phase carrier, and RNA is adsorbed on the carrier;
A method for extracting RNA, comprising: a step of washing the carrier on which A is adsorbed with a washing solution not containing an organic solvent; and a step of eluting RNA adsorbed on the carrier in an eluate, Includes a reverse transcriptase, dNTP, and a primer for reverse transcription. The cleaning liquid may be water or a low salt concentration aqueous solution. The salt concentration of the low salt concentration aqueous solution may be 100 mM or less. The eluate may contain a polymerase and a primer for DNA amplification. The eluate preferably contains BSA. The RNA adsorbed on the carrier may be eluted at 40 ° C. or higher and 70 ° C. or lower. The lysate is 3-7 M guanidine salt, 0-5% nonionic surfactant, 0-0.2 mM EDTA, 0-0.
It may be a neutral solution containing a 2M reducing agent. The carrier may be magnetic particles.

A further embodiment of the invention is a neutral lysate containing 3-7 M guanidine salt, 0-5% nonionic surfactant, 0-0.2 mM EDTA, 0-0.2 M reducing agent. A cleaning solution comprising water or a low salt concentration aqueous solution containing no organic solvent, reverse transcriptase, and dNTP,
And an eluate containing the RNA extraction kit. The eluate may further contain a polymerase.

According to the present invention, it is possible to provide an RNA extraction method that can be used efficiently.

In one Example of this invention, it is a graph which shows the relationship between the number of PCR cycles and DNA amplification amount when examining the amplification product obtained by different RNA elution / RT-PCT method.

Unless otherwise described in the embodiments and examples, MR Green & J. Sambrook (Ed.
), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Pres
s, Cold Spring Harbor, New York (2012); FM Ausubel, R. Brent, RE Kingston,
DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in M
A method described in a standard protocol collection such as olecular Biology, John Wiley & Sons Ltd., or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Reagent for RNA extraction ==
In the method for extracting ribonucleic acid (RNA) according to the present invention, a sample containing RNA is mixed with a lysate containing a chaotropic substance and a nucleic acid-binding solid phase carrier, and the RNA is adsorbed on the carrier (adsorption step). ), Washing the carrier on which RNA is adsorbed with a washing solution not containing an organic solvent (washing step), and eluting the RNA adsorbed on the carrier into an eluate containing reverse transcriptase, dNTP, and primer oligonucleotide. Step (elution step).

The sample from which RNA is extracted is not particularly limited as long as it contains RNA, biological samples such as cells and cell masses such as tissues, viruses, synthetic RNA, samples in which impurities or contaminants are mixed into RNA once isolated. It may be.

A chaotropic substance generates chaotropic ions (monovalent anions having a large ionic radius) in an aqueous solution and has an action of increasing the water solubility of hydrophobic molecules, contributing to adsorption of RNA to a solid phase carrier. If it is a thing, it will not specifically limit. Specific examples include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate and the like. Among these, guanidine thiocyanate or guanidine hydrochloride having a strong protein-denaturing action is used. preferable. The concentration of these chaotropic substances used varies depending on each substance. For example, when guanidine thiocyanate is used, it is in the range of 3 to 5.5M, and when guanidine hydrochloride is used, it is used at 5M or more. Is preferred.

The lysing solution is not particularly limited as long as it contains such a chaotropic substance, but it may contain a surfactant for the purpose of disrupting cell membranes or denaturing proteins contained in cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells or the like. Specifically, a Triton surfactant such as Triton-X or Tw
Nonionic surfactants such as tween surfactants such as een20 and anionic surfactants such as sodium N-lauroyl sarcosine (SDS) can be mentioned. It is preferable to use it in the range of 1 to 2%. Furthermore, it is preferable that the solution contains a reducing agent such as 2-mercaptoethanol or dithiothreitol. The lysis solution may be a buffer solution, but is preferably neutral at pH 6-8. In consideration of these matters, specifically, it contains 3 to 7 M guanidine salt, 0 to 5% nonionic surfactant, 0 to 0.2 mM EDTA, 0 to 0.2 M reducing agent and the like. It is preferable to do.

The nucleic acid-binding solid phase carrier is not particularly limited as long as it is a solid having a hydrophilic surface capable of adsorbing nucleic acid in the presence of chaotropic ions, that is, holding the nucleic acid by reversible physical binding. Specifically, a substance containing silicon dioxide, for example, silica, glass, diatomaceous earth, or those subjected to surface treatment by chemical modification is preferable, and a complex with a magnetic substance or a superparamagnetic metal oxide is used. More preferred. When surface treatment is performed by chemical modification, an appropriate positive charge may be imparted to the extent that reversible binding to nucleic acids is not hindered.

Specific examples of the nucleic acid-binding solid phase include particles, filters, bags, dishes, reaction vessels, and the like, but are not particularly limited. Among these, the particle form is more preferable in consideration of the efficiency of adsorption and elution. The particle size in that case is not particularly limited,
0.05-500 micrometers may be sufficient, Preferably it is 1-100 micrometers, Most preferably, it is 1-10 micrometers.

The washing liquid is substantially free of organic solvents such as ethanol and isopropyl alcohol and chaotropic substances, and is preferably water or a low salt concentration aqueous solution. In the case of a low salt concentration aqueous solution, a buffer solution is preferable. . The salt concentration of the low salt concentration aqueous solution is 100
mM or less is preferred, 50 mM or less is more preferred, and 15 mM or less is most preferred. The lower limit of the low salt concentration aqueous solution is not particularly limited, but is preferably 0.1 mM or more, more preferably 1 mM or more, and most preferably 10 mM or more. Moreover, this solution may contain surfactants, such as Triton, Tween, and SDS, and pH is not specifically limited. The salt for making the buffer is not particularly limited, but Tris, Hepes, Pipes,
A salt such as phosphoric acid is preferably used.

The eluate is not particularly limited as long as it contains reverse transcriptase, dNTP, and reverse transcriptase primer (oligonucleotide). In addition, DNA polymerase and primers (oligonucleotides) for DNA polymerase may be included, and real-time PCR such as TaqMan probe, Molecular Beacon, cycling probe, etc.
And a fluorescent dye for intercalator such as SYBR green. Furthermore, it is preferable to contain BSA (bovine serum albumin) or gelatin as a reaction inhibition inhibitor. The solvent is preferably water, and more preferably an organic solvent such as ethanol or isopropyl alcohol and a substance that does not substantially contain a chaotropic substance. Moreover, it is preferable to contain a salt so that it may become a buffer for reverse transcriptase and / or a buffer for DNA polymerase. The salt for making the buffer solution is not particularly limited as long as it does not inhibit the enzyme reaction, but salts such as Tris, Hepes, Pipes, and phosphoric acid are preferably used. The reverse transcriptase is not particularly limited. For example, Avian Myeloblast virus (Avian Myelo)
blast Virus), Las Associated Virus Type 2 (Ras Associa)
ted Virus type 2), mouse Moloney Muleen Leukemia virus (Mous)
e Molony Murine Leukemia Virus), reverse transcriptase derived from human immunodeficiency virus type 1 (Human Immunodefective Virus type 1), and the like can be used, but a thermostable enzyme is preferred. The DNA polymerase is not particularly limited, but a heat-resistant enzyme or a PCR enzyme is preferable. For example, Taq polymerase, Tfi
There are numerous commercial products such as polymerase, Tth polymerase, or improved versions thereof.

The concentration of dNTP or salt contained in the eluate may be a concentration suitable for the enzyme to be used. Usually, dNTP is 10 to 1000 μM, preferably 100 to 500 μM, Mg ²⁺ is 1 to 100 mM, preferably 5 to 10 mM. , Cl ^{− from 1} to 2000 mM, preferably 20
The total ion concentration is not particularly limited, but may be a concentration higher than 50 mM, preferably a concentration higher than 100 mM, more preferably a concentration higher than 120 mM, and a concentration higher than 150 mM. Preferably, a concentration higher than 200 mM is more preferable. The upper limit is preferably 500 mM or less, more preferably 300 mM or less, and 2
More preferred is 00 mM or less. Each of the primer oligonucleotides is 0.1 to
10 μM, preferably 0.1 to 1 μM is used. The concentration of BSA or gelatin is 1
If it is less than mg / mL, the effect of preventing reaction inhibition is small, and if it is more than 10 mg / mL, reverse transcription reaction and subsequent enzyme reaction may be inhibited, so 1-10 mg / mL is preferable. When gelatin is used, its origin can be exemplified by cow skin, pig skin, and cow bone, but is not particularly limited.
If gelatin is difficult to dissolve, it may be dissolved by heating.

== RNA extraction method ==
Specifically, RNA may be extracted as follows.
First, as a lysis step, a sample for extracting RNA and a nucleic acid-binding solid phase carrier are mixed with an appropriate amount of lysate, and the sample is crushed by a homogenizer, a vortex mixer, or the like.
NA is adsorbed on the carrier.

Next, in order to remove non-specifically adsorbed impurities on the carrier, it is preferable to wash the carrier on which RNA has been adsorbed with an appropriate amount of lysis solution. Although the number of times of washing is not particularly limited, it may be washed once to several times.

The term “washing” as used herein refers to an operation of removing a non-specifically bound substance other than RNA from the carrier by bringing the carrier to which RNA is bound into contact with a washing solution and separating it again. The specific separation method varies depending on the form of the carrier to be used, but when the carrier is in the form of particles such as beads, centrifugation, filtration separation, column operation, and the like can be used. When the carrier contains a magnetic material, the carrier can be easily washed using a magnet or the like.

Then, as an elution step, an appropriate amount of eluate is added to the carrier on which RNA is adsorbed, and mixed by a vortex mixer or the like to elute the RNA from the carrier. At this time, RNA extraction is promoted by heating the eluate. Although heating temperature is not specifically limited, What is necessary is just to be higher than 40 degreeC, 50 degreeC or more is preferable and 60 degreeC or more is more preferable. The upper limit of the heating temperature is not particularly limited, but is preferably 70 ° C. or less, more preferably 65 ° C. or less, further preferably 60 ° C. or less, and most preferably 60 ° C. As a heating method, the carrier may be added to the preheated eluate, or may be heated after the carrier is added to the eluate. The heating time is not particularly limited, but is preferably about 30 seconds to 10 minutes.

Since the eluate contains reverse transcriptase, dNTP, and reverse transcriptase primer, the eluate can be used for the reverse transcription reaction as it is. At this time, the tube used for elution may be used as it is, or may be transferred to a new tube, but it is preferable to use the tube used for elution as it is in view of reaction efficiency. In addition, a part or all of the eluate may be used for the reverse transcription reaction, and when part is used, it is preferably diluted with a buffer adjusted for reverse transcriptase. As the buffer adjusted for reverse transcriptase, a solution having the same components as the eluate may be used, but the salt concentration is preferably adjusted appropriately, and reverse transcriptase, dNTP,
And the primer for reverse transcriptase may or may not be added. At this time, the carrier may or may not be removed, but is preferably removed.

The reverse transcription reaction may be performed under conditions suitable for reverse transcriptase. After the reverse transcription reaction, the amplified cDNA can be used for various purposes such as polymerase reaction including PCR. In the examples, a reverse transcription reaction was performed during the elution step. Thereby, the entire reaction time can be shortened without providing time for the reverse transcription reaction. In that case, the reaction temperature is preferably 60 ° C. or higher and 70 ° C. or lower, more preferably 60 ° C. or higher and 65 ° C. or lower, and 60 ° C. in order to simultaneously proceed with elution of nucleic acid and reverse transcription reaction. Is most preferred.

When the eluate contains DNA polymerase and a primer for DNA polymerase, the reaction solution after the reverse transcription reaction can be directly used for the DNA polymerase reaction. At this time, the tube used for the reverse transcription reaction may be used as it is, or may be transferred to a new tube. However, in terms of reaction efficiency, it is preferable to use the tube used for the reverse transcription reaction as it is. In addition, a part or all of the reaction solution may be used for the DNA polymerase reaction, and when a part is used, it is preferably diluted with a buffer adjusted for DNA polymerase. DNA
As the buffer adjusted for the polymerase, a solution having the same components as the eluate may be used, but the salt concentration is preferably adjusted appropriately, and DNA polymerase, dNTP, and DN are preferably adjusted.
The primer for A polymerase may or may not be added.

The DNA polymerase reaction may be performed under conditions suitable for the DNA polymerase to be used. After the DNA polymerase reaction, the amplified cDNA can be used for various purposes such as library preparation.

== RNA extraction kit ==
The RNA extraction kit of the present invention is a neutral lysate containing 3 to 7 M guanidine salt, 0 to 5% nonionic surfactant, 0 to 0.2 mM EDTA, and 0 to 0.2 M reducing agent. And a washing solution composed of water or a low salt concentration aqueous solution not containing an organic solvent, and an eluate containing reverse transcriptase and dNTP. With this kit, RNA extraction and reverse transcription according to the present invention can be easily performed simply by adding a reverse transcription primer corresponding to the target gene to the eluate.

The kit may further contain a polymerase. Thus, RNA extraction, reverse transcription, and DNA amplification according to the present invention can be easily performed by simply adding a primer for DNA amplification corresponding to the target gene to the eluate.

The components of the kit, such as lysis solution, washing solution and eluate, are the same as those described in the RNA extraction reagent.

(1) Lysing step and washing step To a 150 μL serum sample (collected from a patient suffering from influenza A) in a 1.5 mL Eppendorf microcentrifuge tube, 35
0 μL of a lysis solution (7 M guanidine hydrochloride, 0.15 mM ethylenediaminetetraacetic acid dihydrogen dihydrate (EDTA · 2H ₂ O), 2% Triton-X100, 0.15 M 2-mercaptoethanol) was added and dissolved. To this solution, magnetic silica particles (NPK-401
, Toyobo Co., Ltd.) was added, and vortex mixer (Vortex mix) at room temperature for 5 minutes.
er). Thereafter, the microtube was placed on a magnetic stand (MGS-101, Toyobo Co., Ltd.) to collect magnetic silica particles, and the supernatant was removed.
Next, remove the microtube from the magnetic stand, add 350 μL of Washing Liquid I (7M guanidine hydrochloride), mix well, then place it on the magnetic stand again, and remove the supernatant to wash the magnetic beads. did. Next, 450 μL of washing solution II (5 mM in the same manner)
The particles were washed with (Tris-HCl buffer).

(2) Elution step to reverse transcription reaction solution (reverse transcription) (Sample 1)
Add 20 μL of Eluent I to the particles collected in the washing step, suspend the particles,
After heating to ° C., the microtube was placed on a magnetic stand and the supernatant was collected. During this heating period, RNA is eluted from the particles and a reverse transcription reaction occurs.
2 μL of the supernatant was added to 18 μL of the PCR reaction solution II in the PCR reaction vessel, real-time PCR was performed using the following primers, and the luminance was measured for each cycle.

(3) Elution into PCR reaction solution (Sample 2)
Add 20 μL of Eluate III to the particles collected in the washing step, suspend the particles,
After heating to 60 ° C., the microtube was placed on a magnetic stand and the supernatant was collected.
This supernatant is transferred to a PCR reaction vessel as it is, and real-time P using the following primers:
CR was performed and the brightness was measured for each cycle.

(4) Elution into water (conventional method) (Sample 3)
Purified water treated with 20 μL of DEPC was added to the particles collected in the washing step, the particles were suspended, heated to 60 ° C. for 5 minutes, and then the microtube was placed on a magnetic stand to collect the supernatant. .
2 μL of the supernatant was added to 8 μL of the reverse transcription reaction solution IV and heated to 50 ° C. for 5 minutes to perform the reverse transcription reaction.
Furthermore, 2 μL of the reaction solution was added to 18 μL of the PCR reaction solution V in the PCR reaction vessel, real-time PCR was performed using the following primers, and the luminance was measured for each cycle.

Primer
Primer F: GAC CAA TCC TGT CAC CTC TGA C
Primer R: AGG GCA TTT TGG ACA AAG CGT CTA
probe
TaqMan probe: FAM- TGC AGT CCT CGC TCA CTG GGC ACG -TAMRA

As shown in FIG. 1, the number of rising cycles (Ct value) of the amplification curve in the case of Sample 1 was 1.0 cycle earlier than that in the case of Sample 3. Ct for sample 2
The value was 4.0 cycles earlier than the amplification curve for sample 3.

Thus, RNA can be detected more efficiently than before by the RNA extraction method of the present invention.

Claims (10)

Mixing a lysate containing ribonucleic acid (RNA) with a lysate containing a chaotropic substance and a nucleic acid binding solid phase carrier to adsorb RNA onto the carrier;
Washing the carrier on which the RNA has been adsorbed with a washing solution containing no organic solvent;
Eluting RNA adsorbed on the carrier into an eluate;
A method for extracting RNA comprising
The extraction method, wherein the eluate contains reverse transcriptase, dNTP, and reverse transcription primer. The extraction method according to claim 1, wherein the cleaning liquid is water or a low salt concentration aqueous solution. The extraction method according to claim 2, wherein the salt concentration of the low salt concentration aqueous solution is 100 mM or less. The eluate contains a polymerase and primers for DNA amplification,
The extraction method according to any one of claims 1 to 3. The extraction method according to any one of claims 1 to 4, wherein the eluate contains BSA. The method according to any one of claims 1 to 5, wherein RNA adsorbed on the carrier is eluted at 40 ° C or higher and 70 ° C or lower. The solution is 3-7M guanidine salt, 0-5% nonionic surfactant, 0-0.
The method according to claim 1, which is a neutral lysate containing 2 mM EDTA and 0 to 0.2 M reducing agent. The method according to claim 1, wherein the carrier is magnetic particles. 3-7M guanidine salt, 0-5% nonionic surfactant, 0-0.2 mM EDT
A, a neutral solution containing 0 to 0.2 M reducing agent;
A cleaning solution comprising water or a low salt concentration aqueous solution not containing an organic solvent;
An eluate containing reverse transcriptase and dNTPs;
RNA extraction kit comprising The RNA extraction kit according to claim 9, wherein the eluate further contains a polymerase.