WO2014065395A1 - Rna調製法 - Google Patents
Rna調製法 Download PDFInfo
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- WO2014065395A1 WO2014065395A1 PCT/JP2013/078939 JP2013078939W WO2014065395A1 WO 2014065395 A1 WO2014065395 A1 WO 2014065395A1 JP 2013078939 W JP2013078939 W JP 2013078939W WO 2014065395 A1 WO2014065395 A1 WO 2014065395A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Definitions
- the present invention relates to a method for non-degrading extraction of RNA in a biological sample, which uses an RNA extraction reagent containing an alkali metal salt and a surfactant.
- RNA which is one of the analysis targets in these genetic tests, is unstable compared to DNA, and is easily degraded by endogenous ribonuclease contained in a biological sample or high temperature / alkaline treatment. Therefore, the preparation of RNA required advanced skills to prevent its degradation, multi-step experimental processes, and expensive dedicated equipment and reagents.
- Acid Guanidium Phenol recovers non-degraded RNA by dissolving a specimen by combining a protein denaturant and an organic solvent and inactivating endogenous ribonuclease.
- Chloroform (AGPC) method Non-patent document 1
- hot phenol method Non-patent document 2
- each method includes an organic solvent that inhibits an enzymatic reaction such as a nucleic acid amplification reaction and a high concentration of a denaturing agent, it is not only high in risk but also has a long and multi-step process for removing them. Processing time is required, and there are problems in terms of cost and simplicity.
- RNA is extracted using a strong chaotropic substance and a surfactant as a protein denaturant without using an organic solvent, and the extract is directly subjected to an enzymatic reaction.
- a method is known in which a biological sample is dissolved using guanidine thiocyanate and sarkosyl as denaturing agents and RNA is extracted while protecting it from degradation by endogenous ribonuclease (Patent Document 1). This extract can be directly subjected to an enzymatic reaction.
- RNA can be extracted more easily than the conventional method without requiring a step of removing the denaturant before subjecting the extract to an enzyme reaction.
- strong chaotropic substances such as guanidine thiocyanate and sarkosyl are strong denaturing agents for proteins, and the presence of these strong denaturing agents in an enzyme reaction system is not preferable from the viewpoint of an efficient enzymatic reaction.
- Patent Document 2 a method using cholic acid or glycolic acid (Patent Document 2) is known. According to this method, the nucleic acid extracted from the biological sample can be directly subjected to an enzyme reaction such as a nucleic acid amplification method without requiring a purification step or a dilution step. However, this method could not prevent RNA degradation by endogenous ribonuclease, and extraction of non-degraded RNA was impossible.
- An object of the present invention is to provide a means for preparing RNA that can be used in an enzyme reaction more easily than conventional methods.
- the present invention relates to a reagent for extracting RNA in a biological sample, comprising an alkali metal salt and a surfactant.
- the alkali metal salt is preferably an alkali metal halide.
- the alkali metal salt is preferably lithium chloride.
- the surfactant contains glycolic acids.
- the surfactant further contains deoxycholic acids.
- the present invention also relates to an RNA extraction kit comprising the reagent.
- the present invention also relates to a method for extracting RNA in a biological sample, characterized in that the reagent or RNA extraction kit is used.
- RNA that can be used for enzymes and chemical reactions can be prepared in a non-degraded state more easily than conventional methods.
- FIG. 3 is a diagram showing the results of extracting RNA from a cell and subjecting it to RT-PCR.
- RNA is prepared.
- the biological sample is derived from animal or plant cells, tissues, whole blood, serum, lymph, tissue fluid, urine, semen, vaginal fluid, amniotic fluid, tears, saliva, sweat, and other body fluids, exosomes, and other cells.
- examples include vesicles, feces, sputum, bacteria, viruses and the like, but are not limited to these as long as they contain nucleic acids.
- RNA is exemplified by non-translatable RNA such as messenger RNA, transfer RNA, ribosomal RNA, small nuclear RNA, small nuclear RNA, micro RNA, and the like, but includes a ribonucleotide polymer. It is not limited to this.
- RNA preparation method of the present invention has the advantage that the RNA extract can be directly used for subsequent analysis, and the treatment solution contains all RNAs of molecular weight contained in the sample without loss.
- an RNA extraction reagent for extracting RNA contains an alkali metal salt as an RNA protecting agent.
- Examples of the alkali metal salt in the present invention include lithium salt, sodium salt, potassium salt, rubidium salt, and cesium salt.
- Examples include, but are not limited to, rubidium chloride, rubidium chloride, rubidium bromide, rubidium iodide, rubidium acetate, cesium chloride, cesium bromide, cesium iodide, and cesium
- Alkali metal salts have an RNA protecting effect, but do not serve as cofactors for nucleic acid-related enzymes, and have the advantage of having less influence on enzyme activity than other metal salts that generate polyvalent cations that can be cofactors.
- the concentration of the alkali metal salt contained in the RNA extraction reagent can be easily determined by those skilled in the art, but as an example, it is preferably 0.2 M or more and a saturation concentration or less, preferably 0.3 M or more, and 5. 0M or less is more preferable, and 0.3M or more and 2.5M or less is more preferable. If the concentration of alkali metal salt is too low, RNA cannot be protected from degradation by endogenous ribonuclease, and if the concentration is too high, RNA can be protected, but it tends to inhibit subsequent enzymatic reactions. .
- the RNA extraction reagent contains a surfactant as a sample lysing agent.
- the surfactant in the present invention include ionic, nonionic and amphoteric surfactants.
- Tweens such as Tween 20, Tween 40, Tween 60 and Tween 80, Tritons such as Triton X-100, Triton X-114, Triton XL-80N, nonionic surfactants such as Nonidets and NP-40, and cholic acids
- anionic surfactants such as deoxycholic acids and glycolic acids, more preferably Tween20, Triton X-100, deoxycholic acid and glycolic acid, which are surfactants that do not inhibit the enzyme reaction. If there is, it is not limited to this.
- These surfactants may be used alone or in combination.
- the concentration of the surfactant contained in the RNA extraction reagent can be easily determined by those skilled in the art, but when cholic acids, deoxycholic acids, or glycolic acids are used, the concentration is 1 mM or more. It is preferable. If the concentration of the surfactant is too low, there is a tendency that the biological sample cannot be dissolved.
- the RNA extraction reagent may contain a buffer.
- a buffer such as a phosphate buffer or a Good buffer is preferably used.
- MES Bis-Tris
- ADA PIPES
- ACES MOPSO
- BES MOPS
- TES HEPES
- DIPSO DIPSO
- TAPSO POPSO
- HEPPSO EPPS
- Tricine Tris
- Bicine Tris
- TAPS CHES
- CAPSO CAPS
- CAPS CAPS
- These buffering agents may be used alone or in combination.
- the RNA extraction reagent is a water-soluble protein component such as albumin having an effect of reducing enzyme reaction inhibition by contaminants derived from a biological sample, polyamine, cyclodextrin, trehalose, PVP (Polyvinylpyrrolidone), polyethylene glycol and the like. It may contain a polymer.
- the RNA extraction reagent may contain an antifreeze agent such as glycerol, betaine or sucrose for preventing freezing under freezing.
- an antifreeze agent such as glycerol, betaine or sucrose for preventing freezing under freezing.
- RNA extraction reagents include chelating agents, nuclease inhibitors, reducing agents such as DTT (dithiothreitol) for further ensuring the inactivation of nuclease activity, organics such as DMSO and formamide for subsequent enzymatic reactions.
- a solvent may be included.
- the mixing ratio of the biological sample and the RNA extraction reagent is preferably 9: 1 to 1: 999, more preferably 4: 1 to 1: 499, and further preferably 1: 1 to 1:99. preferable.
- the biological sample after mixing the biological sample and the RNA extraction reagent, the biological sample may be physically crushed during the RNA extraction treatment, for example, combined with crushing by freezing and thawing or physical crushing using a homogenizer. Is possible. Furthermore, according to the present invention, RNA can be extracted without crushing operation before mixing the biological sample and the RNA extraction reagent.
- heat treatment may be performed in order to extract RNA from the biological sample.
- the heat treatment temperature is preferably 0 ° C. or higher and 100 ° C. or lower, more preferably 30 ° C. or higher and 90 ° C. or lower, and further preferably 50 ° C. or higher and 80 ° C. or lower. If the temperature is too low, the extraction efficiency tends to be low, and if it is too high, the RNA tends to be degraded.
- the heat treatment time is preferably 30 minutes or less, more preferably 15 minutes or less. If the treatment time is too short, the extraction efficiency tends to be low, and if it is too long, the RNA tends to be degraded.
- an enzyme reaction such as a nucleic acid amplification reaction by mixing the RNA extract directly into the enzyme reaction solution as a substrate without the need for further purification steps or dilution steps.
- enzymes to be reacted include nucleases such as deoxyribonuclease, ribonuclease, exonuclease and endonuclease, proteases such as proteinases and peptidases, DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and RNA-dependent RNA Examples include polymerases, thermostable polymerases, strand displacement polymerases, polymerases such as terminal transferases, ligases, recombinases, lysozymes, cellulases and the like.
- the mixing ratio of the RNA extract and the enzyme reaction solution is preferably 1: 999 to 999: 1, more preferably 1:99 to 99: 1, and 1:
- the RNA extract can be mixed with a nucleic acid fluorescent labeling reagent such as ethidium bromide, SYBR (R) Green, PicoGreen (R) or a fluorescent labeled probe, and the contained RNA can be detected. It is also possible to subject the RNA extract to a nucleic acid amplification reaction in the presence of a fluorescent labeling reagent and monitor the amplification reaction in real time. It is also possible to subject the RNA extract to a sequence reaction.
- a nucleic acid fluorescent labeling reagent such as ethidium bromide, SYBR (R) Green, PicoGreen (R) or a fluorescent labeled probe
- the nucleic acid amplification reaction is a method of amplifying a nucleic acid sequence represented by the PCR method.
- the LCR Liigase Chain Reaction
- the SDA Strand Displacement Amplification
- the RCA Rolling
- Circle Amplification (CPT) Method Cycling Probe Technology (CPT) Method
- Q-Beta Replicate Amplification Technology Method ICAN (Isothermal and ChimeraimPrincipal Amplification Technology).
- ermal Amplificaton of DNA) method NASBA (Nucleic acid Sequence-based Amplification method) method
- TMA although Transcription mediated amplification method known methods such method is exemplified, but not limited thereto.
- a molecule having specific binding ability such as a molecule having a nucleic acid partially complementary to the target nucleic acid contained in the RNA extract, an antibody, or a single-stranded nucleic acid binding protein is mixed with the target nucleic acid. It is possible to bind specifically. That is, by using the RNA extract, Southern blotting, Northern blotting, real-time PCR, labeling of specific nucleic acid with a labeled nucleic acid probe, detection, purification, separation and the like are possible.
- the RNA extract is subjected to various chromatographies such as ion exchange columns and gel filtration columns, centrifugation, filtration, dialysis, and adsorption treatment to a solid phase carrier to separate impurities, and the RNA extract is contained in the RNA extract. It is also possible to purify RNA. Further, these means can be appropriately combined and used as an RNA purification kit.
- the kit of the present invention has the RNA extraction reagent.
- a sample washing solution deoxyribonuclease, protease, reverse transcriptase, DNA polymerase and its substrate, and oligonucleotide may be included.
- the reagent or kit of the present invention can be incorporated into a nucleic acid preparation device, a nucleic acid amplification device, a nucleic acid automatic analysis device, or the like.
- RNA extraction reagent having the following composition was prepared. Lithium chloride, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, 0.05% Triton X-100
- Lithium chloride is extracted with RNA at concentrations of 0M, 0.1M, 0.2M, 0.5M, 1.0M, 2.0M, 3.0M, 4.0M, 5.0M, 6.0M, 7.0M Reagents were prepared.
- RNA extraction Blood was collected from the mice. Heparin was used as an anticoagulant. 18 ⁇ l of RNA extraction reagent was added to 2 ⁇ l of this mouse blood and incubated at 75 ° C. for 5 minutes. After the incubated solution was cooled to room temperature, 2 ⁇ l of DNase I (amount corresponding to 10 Units) was added and incubated at 42 ° C./5 minutes ⁇ 75 ° C./10 minutes.
- RNA sample prepared above was cooled to room temperature and RT-PCR was performed using it as a template to obtain an amplified fragment of nucleic acid derived from the specimen.
- RT-PCR targeted H3F3A mRNA and used forward primer F1 (5′-GGCCCTACTTGCCCTCCTGCAA-3 ′; SEQ ID NO: 1) and reverse primer R1 (5′-GCAAGAGTCGCCCCCTCTACTG-3 ′; SEQ ID NO: 2) as a primer set.
- RT-PCR was performed using PrimeScript One Step RT-PCR Kit Ver. 2 (manufactured by Takara Bio Inc.) was used by adding 2% volume of the RNA sample prepared above to the reaction solution.
- the reaction consists of a reverse transcription reaction at 50 ° C./30 minutes and a 94 ° C./1 minute reverse transcriptase inactivation treatment followed by a PCR cycle of 94 ° C./15 seconds ⁇ 62 ° C./15 seconds ⁇ 72 ° C./15 seconds. 30 cycles were performed. Also, as a negative control, in order to confirm that the amplified fragment was not derived from DNA, a PCR cycle of 94 ° C./15 seconds ⁇ 60 ° C./15 seconds ⁇ 72 ° C./15 seconds was performed without performing a reverse transcription reaction. For 30 cycles. Next, the RT-PCR product was subjected to agarose gel electrophoresis based on a conventional method to visualize the amplified fragment.
- RNA extraction reagent containing an alkali metal salt having the following composition was prepared. 0.7 M alkali metal salt, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, 0.05% Triton X-100
- Alkali metal salts include lithium chloride, lithium bromide, lithium iodide, sodium chloride, sodium iodide, potassium chloride, potassium iodide, rubidium chloride, cesium chloride, lithium acetate, sodium dihydrogen phosphate, sodium tartrate, sodium nitrate Potassium acetate, dipotassium hydrogen phosphate, and potassium sulfate were used.
- RNA extraction reagent was prepared using lithium chloride as the alkali metal salt and omitting deoxycholic acid as the surfactant.
- RNA extraction Using the prepared RNA extraction reagent, blood was collected from the mouse by the same method as in Example 1, and RNA was extracted.
- RT-PCR In the same manner as in Example 1, RT-PCR was performed using the RNA sample extracted above as a template to obtain amplified fragments of nucleic acids derived from each specimen.
- Table 2 shows the results of examining the presence or absence of amplification visualized by agarose gel electrophoresis.
- RNA extraction reagent containing an alkali metal salt When an RNA extraction reagent containing an alkali metal salt was used, amplified fragments were confirmed only in the case of RT-PCR, indicating that RNA was appropriately extracted. Further, among the alkali metal salts, when an RNA extraction reagent containing an alkali metal halide was used, the amplification efficiency in RT-PCR was high. Further, it was found that these RNA extraction reagents do not inhibit DNase I treatment, RT reaction, and PCR.
- RNA extraction reagent containing a strong chaotropic substance or polyvalent metal salt or ammonium salt having the following composition was prepared. 0.7 M strong chaotropic substance or polyvalent metal salt or ammonium salt, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, 0.05% Triton X-100
- Guanidine thiocyanate, guanidine hydrochloride, and urea were used as strong chaotropic substances.
- Magnesium chloride, calcium chloride, nickel chloride and manganese chloride were used as the polyvalent metal salt.
- ammonium salt diammonium hydrogen phosphate was used.
- an RNA extraction reagent containing no surfactant as an RNA protecting agent and containing only a surfactant was prepared.
- RNA extraction Using the prepared RNA extraction reagent, blood was collected from the mouse by the same method as in Example 1, and RNA was extracted.
- RT-PCR RT-PCR was performed in the same manner as in Example 1 using the RNA sample extracted above as a template.
- the results of the amplified fragments visualized by agarose gel electrophoresis are shown in Table 2.
- the polyvalent metal salt was used, the amplified fragment was not confirmed by RT-PCR or PCR. From this, it was found that the polyvalent metal ion did not function as an RNA protecting agent or inhibited the enzyme reaction. That is, in this system, polyvalent cations are not preferred as additives for RNA extraction reagents.
- a strong chaotropic substance and ammonium salt were used, the amplified fragment was confirmed by RT-PCR and PCR. From this, it was found that DNase I treatment was inhibited by strong chaotropic substances and ammonium salts.
- RNA extraction reagents in this system.
- salt as an RNA protecting agent was not included, RNA was degraded, and the amplified fragment was not confirmed by RT-PCR or PCR.
- RNA extraction reagent not containing a surfactant having the following composition was prepared. 0.7 M lithium chloride, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 50 mM EDTA
- RNA extraction Using the prepared RNA extraction reagent, blood was collected from the mouse by the same method as in Example 1, and RNA was extracted from the specimen.
- RT-PCR RT-PCR was performed in the same manner as in Example 1 using the RNA sample extracted above as a template.
- RNA extraction from cultured cells RNA was extracted from HEK293T cells and Jurkat cells, which are human cultured cells, by the following procedure, and the presence thereof was confirmed by agarose gel electrophoresis.
- RNA extraction reagent containing lithium chloride having the following composition was prepared. 0.7 M lithium chloride, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, 0.05% Triton X-100
- RNA extraction 10 3 to 10 5 cultured cells were centrifuged and collected as a pellet. 18 ⁇ l of RNA extraction reagent was added to the cell pellet and incubated at 75 ° C. for 5 minutes. After the incubated solution was cooled to room temperature, 2 ⁇ l of DNase I (10 Units) was added and incubated at 42 ° C./5 minutes ⁇ 75 ° C./10 minutes.
- RT-PCR The RNA sample extracted above was cooled to room temperature, and RT-PCR was performed using each as a template to obtain amplified fragments of nucleic acids derived from cells.
- RT-PCR targeted ACTB mRNA and used forward primer F2 (5′-AGATGGCCACGGCTTGCT-3 ′; SEQ ID NO: 3) and reverse primer R2 (5′-AACCGCCTCATTGCCAATGG-3 ′; SEQ ID NO: 4) as a primer set.
- RT-PCR was performed using PrimeScript One Step RT-PCR Kit Ver. 2 (manufactured by Takara Bio Inc.) was used by adding 2% volume of the RNA sample prepared above to the reaction solution.
- the reaction consists of a reverse transcription reaction of 50 ° C./30 minutes, a 94 ° C./1 minute reverse transcriptase inactivation treatment, and then a PCR cycle of 94 ° C./15 seconds ⁇ 60 ° C./15 seconds ⁇ 72 ° C./15 seconds 30 cycles were performed. Further, as a negative control, PCR cycles of 94 ° C./15 seconds ⁇ 60 ° C./15 seconds ⁇ 72 ° C./15 seconds were performed 30 times without performing a reverse transcription reaction. Next, the RT-PCR product was subjected to agarose gel electrophoresis based on a conventional method to visualize the amplified fragment.
- the amplified fragment visualized by agarose gel electrophoresis is shown in FIG.
- HEK293T cells and Jurkat cells were used as samples, specific amplified fragments were confirmed in both cases, and it was found that RNA was appropriately extracted.
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Abstract
Description
本発明において、生物試料から非分解のRNAを調製するために、アルカリ金属塩と界面活性剤を使用し、検体に内在するリボヌクレアーゼによるRNAの分解を防止しながら、その後の酵素反応に供試可能なRNAを調製する。
マウス血液から、以下の手順でRNAを抽出し、抽出したRNAを鋳型としてRT-PCRを行い、RNAに由来する増幅断片をアガロースゲル電気泳動により確認した。
以下の組成のRNA抽出試薬を調製した。
塩化リチウム、75mM TAPS(pH8.0)、2.25mM CaCl2、15mM MgCl2、175mM グリコール酸、5mM デオキシコール酸、50mM EDTA、0.05% Triton X-100
マウスより血液を回収した。抗凝固剤としてはヘパリンを用いた。このマウス血液2μlにRNA抽出試薬を18μl添加し、75℃にて5分間インキュベートした。インキュベート後の溶液を室温まで冷却した後、2μlのDNase I(10Unitsに相当する量)を添加し、42℃/5分間→75℃/10分間インキュベートした。
上記にて調製したRNA試料を室温まで冷却し、それを鋳型としてRT-PCRを行い、検体に由来する核酸の増幅断片を得た。RT-PCRは、H3F3A mRNAを標的とし、フォワードプライマーF1(5’-GGCCTCACTTGCCTCCTGCAA-3’;配列番号1)、リバースプライマーR1(5’-GCAAGAGTGCGCCCTCTACTG-3’;配列番号2)をプライマーセットとして用いた。RT-PCRはPrimeScript One Step RT-PCR Kit Ver.2(タカラバイオ社製)を用い、上記で調製したRNA試料を反応溶液に対して2%容量添加し行った。反応は、50℃/30分間の逆転写反応、94℃/1分間の逆転写酵素の失活処理の後、94℃/15秒間→62℃/15秒間→72℃/15秒間のPCRサイクルを30サイクル行った。また、ネガティブコントロールとして増幅断片がDNAに由来するものではないことを確認するために、逆転写反応をせずに、94℃/15秒間→60℃/15秒間→72℃/15秒間のPCRサイクルを30サイクル行った。次に、RT-PCR産物を常法に基づきアガロースゲル電気泳動に供し、増幅断片を可視化した。
マウス血液から、以下の手順でRNAを抽出し、抽出したRNAを鋳型としてRT-PCRを行い、RNAに由来する増幅断片をアガロースゲル電気泳動により確認した。
以下の組成のアルカリ金属塩を含むRNA抽出試薬を調製した。
0.7M アルカリ金属塩、75mM TAPS(pH8.0)、2.25mM CaCl2、15mM MgCl2、175mM グリコール酸、5mM デオキシコール酸、50mM EDTA、0.05% Triton X-100
調製したRNA抽出試薬を用いて、実施例1と同様の手法により、マウスより血液を回収し、RNAを抽出した。
実施例1と同様の手法により、上記にて抽出したRNA試料を鋳型としてRT-PCRを行い、各検体に由来する核酸の増幅断片を得た。
マウス血液から、以下の手順でRNAを抽出し、抽出したRNAを鋳型としてRT-PCRを行い、増幅断片をアガロースゲル電気泳動により確認した。
以下の組成の強カオトロピック物質もしくは多価金属塩もしくはアンモニウム塩を含むRNA抽出試薬を調製した。
0.7 M強カオトロピック物質もしくは多価金属塩もしくはアンモニウム塩、75mM TAPS(pH8.0)、2.25mM CaCl2、15mM MgCl2、175mM グリコール酸、5mM デオキシコール酸、50mM EDTA、0.05% Triton X-100
調製したRNA抽出試薬を用いて、実施例1と同様の手法により、マウスより血液を回収し、RNAを抽出した。
実施例1と同様の手法により、上記にて抽出したRNA試料を鋳型としてRT-PCRを行った。
マウス血液から、以下の手順でRNAを抽出し、抽出したRNAを鋳型としてRT-PCRを行い、増幅断片をアガロースゲル電気泳動により確認した。
以下の組成の界面活性剤を含まないRNA抽出試薬を調製した。
0.7M 塩化リチウム、75mM TAPS(pH8.0)、2.25mM CaCl2、15mM MgCl2、50mM EDTA
調製したRNA抽出試薬を用いて、実施例1と同様の手法により、マウスより血液を回収し、検体からRNAを抽出した。
実施例1と同様の手法により、上記にて抽出したRNA試料を鋳型としてRT-PCRを行った。
ヒト培養細胞であるHEK293T細胞及びJurkat細胞から、以下の手順でRNAを抽出し、その存在をアガロースゲル電気泳動により確認した。
以下の組成の塩化リチウムを含むRNA抽出試薬を調製した。
0.7M 塩化リチウム、75mM TAPS(pH8.0)、2.25mM CaCl2、15mM MgCl2、175mM グリコール酸、5mM デオキシコール酸、50mM EDTA、0.05% Triton X-100
103~105個分の培養細胞を遠心分離し、ペレットとして回収した。この細胞ペレットにRNA抽出試薬を18μl添加し、75℃にて5分間インキュベートした。インキュベート後の溶液を室温まで冷却した後、2μlのDNase I(10 Units分)を添加し、42℃/5分間→75℃/10分間インキュベートした。
上記にて抽出したRNA試料を室温まで冷却し、それぞれを鋳型としてRT-PCRを行い、細胞に由来する核酸の増幅断片を得た。RT-PCRは、ACTB mRNAを標的とし、フォワードプライマーF2(5’-AGATGGCCACGGCTGCT-3’;配列番号3)、リバースプライマーR2(5’-AACCGCTCATTGCCAATGG-3’;配列番号4)をプライマーセットとして用いた。RT-PCRはPrimeScript One Step RT-PCR Kit Ver.2(タカラバイオ社製)を用い、上記で調製したRNA試料を反応溶液に対して2%容量添加し行った。反応は、50℃/30分間の逆転写反応、94℃/1分間の逆転写酵素の失活処理の後、94℃/15秒間→60℃/15秒間→72℃/15秒間のPCRサイクルを30サイクル行った。また、ネガティブコントロールとして、逆転写反応をせずに、94℃/15秒間→60℃/15秒間→72℃/15秒間のPCRサイクルを30サイクル行った。次に、RT-PCR産物を常法に基づきアガロースゲル電気泳動に供し、増幅断片を可視化した。
Claims (7)
- アルカリ金属塩及び界面活性剤を含むことを特徴とする、生物試料中のRNAを抽出するための試薬。
- アルカリ金属塩が、アルカリ金属ハロゲン化物である、請求項1に記載の試薬。
- アルカリ金属塩が、塩化リチウムである、請求項1又は2に記載の試薬。
- 界面活性剤が、グリコール酸類を含む、請求項1~3のいずれかに記載の試薬。
- 界面活性剤が、さらに、デオキシコール酸類を含む、請求項4に記載の試薬。
- 請求項1~5のいずれかに記載の試薬を有することを特徴とする、RNA抽出キット。
- 請求項1~5のいずれかに記載の試薬、又は請求項6に記載のRNA抽出キットを用いることを特徴とする、生物試料中のRNAを抽出する方法。
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