US20130144049A1 - Solution for extraction of rna - Google Patents

Solution for extraction of rna Download PDF

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Publication number
US20130144049A1
US20130144049A1 US13/816,792 US201113816792A US2013144049A1 US 20130144049 A1 US20130144049 A1 US 20130144049A1 US 201113816792 A US201113816792 A US 201113816792A US 2013144049 A1 US2013144049 A1 US 2013144049A1
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solution
rna
concentration
biological sample
total amount
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Makiko Yoshimoto
Hideo Akiyama
Hitoshi Nobumasa
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Toray Industries Inc
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Toray Industries Inc
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Assigned to TORAY INDUSTRIES, INC. reassignment TORAY INDUSTRIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AKIYAMA, HIDEO, NOBUMASA, HITOSHI, YOSHIMOTO, MAKIKO
Priority to US15/581,344 priority Critical patent/US10647978B2/en
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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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01C—AMMONIA; CYANOGEN; COMPOUNDS THEREOF
    • C01C3/00—Cyanogen; Compounds thereof
    • C01C3/20—Thiocyanic acid; Salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
    • C07C39/04—Phenol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2866—Grinding or homogeneising

Definitions

  • the present invention relates to a solution for extracting substantially pure RNA from a biological sample.
  • RNA Ribonucleic acid
  • Patent Document 1 discloses a solution for RNA extraction comprising 2 to 5 M guanidine and 40 to 60% phenol. RNA extraction had required not less than 2 days of operation using an ultracentrifuge before, but use of this solution enabled efficient extraction of RNA in 3 hours. This method is called the single-step method.
  • Patent Document 2 discloses an extraction solution for simultaneous extraction and separation of RNA, DNA and proteins from a sample comprising of these components. More specifically, the literature describes extraction and separation of RNA into an aqueous layer by using a 30 to 50% phenol solution containing 0.5 to 2 M guanidine.
  • RNA can be extracted by similar operations using the solutions. That is, each solution is used for homogenization of a biological tissue, and a hydrophobic organic solvent such as chloroform is used upon centrifugation of the homogenate to achieve layer separation. Thereafter, the aqueous layer in the uppermost part comprising RNA is recovered. RNA is then precipitated with alcohol and washed in order to extract RNA.
  • a hydrophobic organic solvent such as chloroform
  • RNA isolated using the solutions and the methods described in Patent Document 1 and 2 still shows contamination with (residual) genomic DNA in an amount which can be detected by the reverse transcription-polymerase chain reaction assay (RT-PCR), leading to problems such as loss of quantitativeness of RNA in cases of RT-PCR (Patent Document 3, e.g., paragraph 0005). Therefore, RNA isolated by these methods needs to be further purified for removal of DNA as a contaminant.
  • RT-PCR reverse transcription-polymerase chain reaction assay
  • a commonly used method for removal of DNA contained as an impurity in an extracted RNA sample is treatment of the RNA sample with deoxyribonuclease (DNase).
  • DNase deoxyribonuclease
  • the extraction is performed using a combination of silica membrane columns, the operation of washing the columns needs to be carried out repeatedly.
  • contamination with DNA is reduced by this treatment with DNase, such additional labor is required and loss of RNA occurs, resulting in a decreased amount of extracted RNA, which is problematic.
  • Patent Document 3 reports a method using an RNA extraction reagent at a pH of less than 4.
  • nucleic acid is depurinated and degraded under acidic conditions, and it is therefore difficult to isolate substantially intact RNA.
  • the solution equilibrium of DNA into the aqueous/organic layer under acidic conditions is biased toward distribution into the organic layer, the effect of suppressing contamination of the aqueous phase with genomic DNA can be expected to some extent by using a reagent for extraction of RNA at a pH of less than 4, but complete suppression of contamination with small DNA fragments having small numbers of bases is impossible.
  • RNA free from contamination with DNA cannot be extracted with conventional solutions for extraction of RNA from biological samples even in cases where quantitativeness is required, which has been problematic. Therefore, for removal of DNA as a contaminant, an additional step such as DNase treatment has been necessary.
  • the present invention aims to solve these problems and provides a solution for extracting substantially pure RNA from a biological sample.
  • the present inventors studied compositions of conventional solutions for RNA extraction and discovered that the phenol concentration has an especially strong relationship with the effect of prevention of contamination with DNA, thereby completing the present invention.
  • the present invention provides the following:
  • RNA can be obtained without an additional treatment such as DNase treatment which may cause recovery loss, which RNA has purity that allows use of the RNA as it is even in uses wherein quantitativeness is required.
  • an RNA of interest can be extracted with high purity even from, among biological samples, body fluids such as blood containing very large amounts of RNase and other contaminants.
  • FIG. 1 shows electropherograms of nucleic acid extracted from serum in Example 1 using a solution according to an embodiment of the present invention.
  • FIG. 2 shows electropherograms of nucleic acid extracted from serum in Comparative Example 1 using a solution described in Patent Document 2.
  • FIG. 3 shows an electropherogram of nucleic acid extracted from serum in Comparative Example 2 using a solution described in Patent Document 1.
  • FIG. 4 shows electropherograms of nucleic acid extracted from serum in Examples 2 to 5 using solutions according to embodiments of the present invention.
  • FIG. 5 shows electropherograms of nucleic acid extracted from serum in Examples 6 to 12 using solutions according to embodiments of the present invention.
  • FIG. 6 shows electropherograms of nucleic acid extracted from serum in Example 13 and Comparative Example 3 using a solution according to an embodiment of the present invention and a solution described in Patent Document 3.
  • FIG. 7 shows electropherograms of nucleic acid extracted from cultured cells in Example 14 using a solution according to an embodiment of the present invention.
  • FIG. 8 shows electropherograms of nucleic acid extracted from serum in Examples 15 and 16 using solutions according to embodiments of the present invention.
  • FIG. 9 shows electropherograms of nucleic acid extracted from serum in Comparative Example 4 using a solution described in Patent Document 3.
  • FIG. 10 shows electropherograms of nucleic acid extracted from serum in Examples 17 and 18 using solutions according to embodiments of the present invention.
  • the present invention provides a solution for extracting RNA from a biological sample, which solution comprises as its components the following (a) to (e):
  • the biological sample used in the present invention comprises RNA and at least DNA. Further, by using the solution of the present invention, substantially pure RNA can be extracted from the biological sample.
  • substantially pure RNA herein means RNA from which DNA contained in the original biological sample has been separated and which is substantially free from contamination with the DNA. Whether or not RNA is substantially pure can be judged by seeing whether or not DNA is detected by electrophoresis, For example, since “Agilent RNA 6000 pico kit” manufactured by Agilent Technologies Inc. (model number, 5067-1513) can be used for detection of nucleic acid in an amount of 50 pg/ ⁇ L to 5000 pg/ ⁇ L (recommendation), the kit can be used for evaluation of the presence/absence of DNA contamination.
  • the extracted nucleic acid may be treated with RNase and subjected to electrophoresis using “Agilent RNA 6000 pico kit”. In cases where no peak was detected, it can be said that DNA contamination was sufficiently suppressed and substantially pure RNA could be obtained. Further, by analyzing the amount of DNA contamination by quantitative PCR, the purity of RNA can be evaluated. For example, in cases where a real-time PCR apparatus and “SYBR Green” (fluorescent dye) are used, double-stranded DNA in an amount of 60 pg can be detected, so that the evaluation can be carried out using these.
  • SYBR Green fluorescent dye
  • extracted nucleic acid is added to a PCR reaction solution containing primers, DNA polymerase and “SYBR Green” to perform PCR amplification, and the result is compared with a preliminarily prepared calibration curve.
  • the amount of DNA contamination can be quantitatively analyzed.
  • the total amount of a solution means the total volume comprising all of the above-described (a) to (e).
  • phenol in an amount of more than 50% by volume based on the total amount of the solution means that more than 500 mL of phenol is contained in 1 L of the solution after mixing of all components.
  • a guanidinium salt at a concentration of 0.5 to 2.0 M based on the total amount of the solution means that the final concentration in the solution is not less than 0.5 M and not more than 2.0 M, that is, the guanidinium salt is contained in an amount of not less than 0.5 mol and not more than 2 mol in 1 L of the solution after mixing of all components.
  • the solution according to an embodiment of the present invention comprises (a) phenol in an amount of more than 50% by volume based on the total amount of the solution. It was found that employing a phenol concentration of more than 50% by volume, which is different from the concentration employed in conventional techniques, produces the effect of reducing contamination of the aqueous layer, into which RNA is extracted, with DNA as an impurity.
  • the solution according to an embodiment of the present invention comprises phenol in an amount of not less than 51% by volume, not less than 52% by volume, not less than 53% by volume, not less than 54% by volume or not less than 55% by volume.
  • the solution of the present invention comprises phenol in an amount of preferably not less than 53% by volume, more preferably not less than 55% by volume.
  • the concentration of phenol is preferably not more than 75% by volume in view of preparing the solution of the present invention in the state where other components of the solution of the present invention, (b) polyalcohol, (c) 0.5 to 2.0 M guanidinium salt and (d) 0.1 to 0.5 M thiocyanate are uniformly mixed at the respective predetermined concentrations. Further, the concentration of phenol is more preferably not more than 65% by volume in view of reducing the influence of oxidation of phenol.
  • the range of the phenol concentration is preferably one determined by an arbitrary combination of these upper limits and lower limits, and is more preferably not less than 52% by volume and not more than 65% by volume, not less than 53% by volume and not more than 65% by volume, especially preferably not less than 55% by volume and not more than 65% by volume.
  • the solution according to an embodiment of the present invention comprises (b) a polyol in an amount of 3 to 10% by volume based on the total amount of the solution.
  • the polyol in the present invention may be an aliphatic alcohol having a plurality of hydroxyl groups, which allows mixing of the (a) phenol component and the aqueous solutions of (c) and (d) in the solution of the present invention, to keep the solution of the present invention uniform.
  • a C 2 -C 6 aliphatic alcohol having 2 to 4 hydroxyl groups is preferred.
  • the polyol include glycerol, ethylene glycol, propylene glycol and erythritol, and the polyol is more preferably glycerol.
  • the polyol may be used in an amount of 3 to 10% by volume based on the total amount of the solution of the present invention in order to maintain the solution of the present invention as a uniform solution and to prevent excessive distribution of the phenol component into the aqueous layer.
  • the solution according to an embodiment of the present invention comprises (c) a guanidinium salt at a concentration of 0.5 to 2.0 M based on the total amount of the solution.
  • a guanidinium salt include guanidinium thiocyanate and guanidinium hydrochloride.
  • Guanidinium salts have an effect to protect RNA from degradation and to keep phenol in the solution state in an aqueous solution.
  • the solution according to an embodiment of the present invention comprises (d) a thiocyanate at a concentration of 0.1 to 0.5 M based on the total amount of the solution.
  • a thiocyanate an inorganic salt of thiocyanic acid may be preferably used, and ammonium thiocyanate and sodium thiocyanate may be more preferably used.
  • the thiocyanate may be a mixture of a plurality of different inorganic salts of thiocyanic acid, and, for example, a mixture of ammonium thiocyanate and sodium thiocyanate may be preferably used.
  • Thiocyanate is considered to enhance RNA extraction from a biological sample.
  • the concentration of guanidinium thiocyanate is included in the concentration of the above-described guanidinium salt, and not included in the concentration of thiocyanate.
  • the solution according to an embodiment of the present invention comprises (e) a buffer for maintaining the pH of the solution at 4 to 6.
  • a buffer for maintaining the pH of the solution at 4 to 6.
  • organic salts and inorganic salts which are conventionally used for maintaining the pH within a desired range and show buffering capacity may be used.
  • Specific examples of the buffer include organic salts and inorganic salts, such as phosphate, acetate, citrate, phthalate, tartrate and lactate, of sodium, potassium, lithium and ammonium.
  • sodium acetate and sodium citrate are more preferably used.
  • a plurality of these organic salts and/or inorganic salts may be used in combination.
  • the concentration of the buffer is not restricted as long as it is sufficient for maintaining the pH within the desired range of 4 to 6, and the concentration is preferably 0.02 to 0.2 M based on the total amount of the solution of the present invention.
  • an appropriate aqueous acid or alkaline solution such as a hydrochloric acid or sodium hydroxide solution may be added as appropriate in addition to the buffer.
  • the solution of the present invention may contain a surfactant(s) such as polyoxyethylene sorbitan, sodium dodecyl sulfate and/or sarcosine for supporting purification of the RNA of interest by denaturing proteins in the biological sample. Further, the solution of the present invention may contain an antioxidant(s) such as hindered amine phenol and/or quinoline for prevention of oxidation of phenol.
  • a surfactant(s) such as polyoxyethylene sorbitan, sodium dodecyl sulfate and/or sarcosine for supporting purification of the RNA of interest by denaturing proteins in the biological sample.
  • an antioxidant(s) such as hindered amine phenol and/or quinoline for prevention of oxidation of phenol.
  • the solution of the present invention may be used in an amount of not less than 1 volume, preferably not less than 3 volumes of the sample.
  • the biological sample is homogenized in the solution of the present invention to form a homogenate.
  • the method of homogenization is not restricted, and examples of the method include stirring by vortexing or the like, crushing with an injection needle or the like, and use of a conventional homogenizer.
  • an organic solvent is added to the homogenate for separation of the aqueous layer, and the resulting mixture is subjected to centrifugation.
  • the organic solvent to be added in this step is preferably used in an amount of about 2% by volume to about 40% by volume based on the homogenate.
  • the centrifugation may be carried out usually at 6,000 ⁇ G to 20,000 ⁇ G for 3 minutes to 30 minutes, for example, at a rate of 12,000 ⁇ G for 10 minutes at room temperature.
  • the rate, temperature and time are not restricted as long as the aqueous layer can be separated.
  • the centrifugation the substantially pure RNA of interest is extracted into the aqueous layer.
  • DNA, proteins and the like are separated into the organic layer, or, in cases where an intermediate layer was produced, DNA, proteins and the like are separated into the organic layer and the intermediate layer.
  • the organic solvent for separation of the aqueous layer is a liquid organic compound to be used for achieving separation into the aqueous layer comprising the RNA of interest extracted using the solution of the present invention and the organic layer and/or the intermediate layer (if produced) comprising DNA and the like.
  • this organic solvent one which has the same degree of hydrophilicity as, or is more hydrophobic than, phenol may be used.
  • CLogP water/octanol distribution coefficient
  • an organic compound having a value of not less than 1.4 CLogP value for phenol
  • an organic compound having a value within the range of 1.4 to 5 may be preferably used.
  • An estimated value of the CLogP value can be calculated by, for example, using a program such as “Chem Draw” (registered trademark).
  • the organic solvent which may be used in the present invention include, but are not limited to, chloroform (1.952), p-bromoanisole (3.064), 1-bromo-3-chloropropane (1.847), 4-bromoveratrole (2.7345), 6-bromo-1,4-benzodioxane (3.0005), 1-bromo-4-trifluoromethoxybenzene (4.173), 1-bromo-2,4-dimethoxybenzene (2.8545), 4-fluoroanisole (2.344), 4-bromotoluene (3.504) and ethyl 4-bromobutyrate (1.772).
  • the value in the parentheses for each of the above organic solvents indicates the CLogP value calculated with “Chem Draw”.
  • the organic solvent for separation of the aqueous layer may be used by formation using the solution of the present invention comprising (a) to (e) as described above and addition to the homogenate, but the organic solvent may also be preliminarily contained in the solution of the present invention comprising the above-described (a) to (e).
  • inclusion of this organic solvent in advance causes separation of the solution into the aqueous layer and the organic layer before mixing with a biological sample, so that it has been difficult to use the solution as an extraction solution.
  • the organic solvent can be uniformly mixed with the solution of the present invention, and the resulting solution can be stored as a single solution.
  • the content of the organic solvent may be selected depending on the type of the organic solvent to be added and the phenol concentration in the solution, within the range in which the organic solvent can be uniformly mixed in the solution of the present invention.
  • chloroform is contained preferably in an arbitrary volume of up to 27% by volume based on the total amount, 100%, of the solution comprising the above-described (a) to (e).
  • chloroform in an arbitrary volume of up to 27 mL, to 100 mL of the solution comprising the above-described (a) to (e).
  • Chloroform is contained in an amount of more preferably 5 to 25% by volume, still more preferably 10 to 20% by volume based on the total amount, 100%, of the solution comprising the above-described (a) to (e).
  • chloroform is contained preferably in an arbitrary volume of up to 14%, more preferably in an amount of 6 to 13% by volume, still more preferably in an amount of 8 to 12% by volume based on the total amount, 100%, of the solution comprising the above-described (a) to (e).
  • p-bromoanisole is contained preferably in an arbitrary volume of up to 22% by volume, more preferably in an amount of 5 to 20% by volume, still more preferably in an amount of 10 to 18% by volume based on the total amount, 100%, of the solution comprising the above-described (a) to (e).
  • p-bromoanisole is contained preferably in an arbitrary volume of up to 13% by volume, more preferably in an amount of 3 to 11% by volume, still more preferably in an amount of 5 to 9% by volume based on the total amount, 100%, of the solution comprising the above-described (a) to (e).
  • a lower alcohol may be added to the aqueous layer comprising RNA in order to precipitate the RNA, and the precipitated RNA may be recovered.
  • the RNA precipitated by addition of a lower alcohol to the aqueous layer comprising RNA may be adsorbed to a carrier to which RNA can be adsorbed, such as a silica membrane column, and the RNA may then be eluted and recovered from the carrier (column).
  • the lower alcohol to be used in this step include ethanol and isopropanol.
  • the concentration of the lower alcohol may be determined according to those employed in conventional techniques such as ethanol precipitation and isopropanol precipitation, or according to the concentrations recommended by manufacturers of carriers such as silica membrane columns.
  • the solution of the present invention can be produced by mixing the above-described (a) to (e) such that their respective concentrations are attained.
  • the procedure of the mixing is not restricted.
  • the respective solutions at higher concentrations may be prepared in advance before mixing the solutions.
  • 6 M aqueous guanidinium thiocyanate solution, 6 M aqueous ammonium thiocyanate solution and 1 M sodium acetate may be prepared in advance and then mixed to attain the concentrations of interest, followed by addition of glycerol, phenol and a necessary amount of water thereto, to prepare the solution of the present invention.
  • the solution of the present invention wherein an organic solvent for separation of the aqueous layer is preliminarily contained in the solution comprising the above-described (a) to (e) can also be similarly produced by mixing (a) to (e) and the organic solvent such that their desired concentrations are attained.
  • the biological sample to be used in the present invention is not restricted as long as it comprises RNA and at least DNA.
  • the biological sample may comprise, in addition to DNA, proteins as impurity components which are preferably separated from the RNA of interest.
  • Specific examples of the biological sample include cultured cells; culture liquids of cultured cells; body tissues such as surgical sections and biopsy samples; living cells; blood; blood components (serum, plasma); urine; and body fluids such as saliva and tears.
  • the biological sample is not restricted to these, and an arbitrary sample containing RNA may be used.
  • the collected sample may be mixed as it is with the solution of the present invention or may be diluted with PBS or water before mixing with the solution of the present invention.
  • the collected sample may be mixed as it is with the solution of the present invention or may be diluted with PBS or water before mixing with the solution of the present invention, and, in cases where the sample is diluted, a homogenate of the biological sample is preferably prepared before dilution with water or PBS in order to prevent degradation of RNA.
  • the solution of the present invention enables effective extraction of contaminants such as proteins into an organic layer, so that the RNA of interest can be obtained with high purity. Further, the intermediate layer that appears after centrifugation is reduced and clear separation into layers can be achieved, so that the aqueous layer comprising the RNA of interest can be easily separated.
  • RNA extracted using the solution is ribonucleic acid wherein a plurality of ribonucleotides are linked by phosphodiester bonds, and the molecular weight, the number of bases and the origin of the RNA are not restricted.
  • RNA is classified into many types according to functional classification, and examples of the types include mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), ncRNA (non-coding RNA), snRNA (small nuclear RNA) and snoRNA (small nucleolar RNA).
  • the molecular weight number of bases
  • RNA having any molecular weight is included in the present invention.
  • RNAs having base numbers of about 15 to 500 bases which are generally called small RNAs
  • RNAs generally having base numbers of about 18 to 25, which are miRNAs (microRNAs) are also included in the RNA of the present invention.
  • RNA having a relatively small number of bases such as small RNA can also be extracted with high purity.
  • RNA and RNA coexist In the state where DNA and RNA coexist, it is usually difficult to distinguish between these and to quantify each of these using an absorptiometer or luminometer.
  • substantially pure RNA can be obtained, so that quantification of RNA using an absorptiometer or luminometer is possible.
  • the solution of the present invention is used in RNA analysis using qRT-PCR or a microarray, the analysis can be simply carried out without requirement of treatment with DNase, in the absence of the noise due to coexistence of DNA.
  • the respective components of the solution were mixed such that their final concentrations were as described below, to prepare a solution for RNA extraction.
  • RNA extraction As a biological sample containing RNA as well as DNA and proteins, serum was used for RNA extraction. By mixing 900 ⁇ L of the solution prepared in the above (1) and 300 ⁇ L of serum by vortexing, the sample was homogenized. To the resulting homogenate, 60 ⁇ L of p-bromoanisole was added, and the resulting mixture was mixed, followed by centrifuging the mixture at room temperature at 12,000 ⁇ G for 10 minutes. By this, an aqueous layer containing RNA, and an organic layer and an intermediate layer containing DNA and proteins were formed. From these, 4004 of the aqueous layer was separated into another tube.
  • RNA separated in (2) 1.5 volumes of 100% ethanol was added, and 700 ⁇ L of the resulting mixture was placed in a column for purification of nucleic acid, “RNeasy Mini Spin Column” contained in “miRNeasy mini kit” (manufactured by QIAGEN), followed by centrifuging the column at 8,000 ⁇ G for 15 seconds to allow adsorption of nucleic acid to the column. The liquid that passed through the column was discarded. By repeating this operation until no ethanol-mixed RNA sample is remaining, all nucleic acid contained in the aqueous layer was adsorbed to the column.
  • the column was washed twice with 700 ⁇ L of Buffer RWT and 500 ⁇ L of Buffer RPE, and the column was then dried, followed by elution with 30 ⁇ L of RNase-free water, to obtain a purified and concentrated RNA sample.
  • the sample separated in (2) was subjected to RNase treatment.
  • RNase treatment To the aqueous layer containing RNA separated in (2), 1.5 volumes of 100% ethanol was added, and nucleic acid was allowed to adsorb to the column in the same manner as in (3a).
  • diluted RNase was added thereto to perform RNase treatment of the nucleic acid adsorbed to the column, and the column was washed twice with 350 ⁇ L of Buffer RWT and 500 ⁇ L of Buffer RPE, followed by drying the column. Thereafter, elution was carried out with 30 ⁇ L of RNase-free water, to obtain a purified and concentrated RNA sample.
  • each sample was rapidly cooled.
  • the samples were then subjected to electrophoresis using “Agilent RNA 6000 pico kit” manufactured by Agilent Technologies Inc. (model number, 5067-1513). The results are shown in FIG. 1 . Further, by the Smear Analysis function of “Bioanalyzer 2100”, the peak area of 25 to 500 nt was calculated to confirm the peak size and the amount (concentration) of nucleic acid detected.
  • the amount of nucleic acid calculated in this case was 63 pg/ ⁇ L, the amount of nucleic acid calculated for lane 2 was considered to be due to the noise. From the above results, the extracted nucleic acid could be confirmed to be RNA which does not contain DNA. Since RNA having 22 to 25 bases and the peak obtained in the present Example showed similar migration distances in electrophoresis, the RNA found in lane 1 was considered to have 22 to 25 bases.
  • Patent Document 2 The solution described in Patent Document 2 was prepared with the same composition as in Example 1 except that the phenol concentration was 50% by volume in terms of the final concentration of the solution.
  • Example 1 The operation was carried out in the same manner as in (3b) in Example 1 except that the aqueous layer containing RNA was treated with DNase instead of the RNase in (3b), to obtain a purified and concentrated sample. Other conditions were the same as in Example 1.
  • Example 2 The operation was carried out in the same manner as in Example 1. The results are shown in FIG. 2 .
  • the same solution as the extraction solution described in Patent Document 1 was prepared except that the phenol concentration was 60% by volume. That is, the solution contained 60% by volume of phenol, 2M guanidinium thiocyanate, 0.1 M sodium acetate and 0.2% by volume of 2-mercaptoethanol in terms of the final concentrations, and the pH of the solution was 4.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in FIG. 3 .
  • a solution was prepared such that the composition of the solution is the same as in Example 1 except that the phenol concentration was 55% by volume in terms of the final concentration.
  • Example 1 The same peak as in Example 1 was detected (lane 1 ), and it could be confirmed that only RNA was extracted with high purity. Since the peak in the RNase-treated sample (lane 5 ; RNase (+)) was as weak as that in BLANK, it could be confirmed that the extracted nucleic acid contained only RNA.
  • a solution was prepared such that the composition of the solution is the same as in Example 1 except that the phenol concentration was 65% by volume in terms of the final concentration.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 2 in FIG. 4 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • a solution was prepared such that the composition of the solution is the same as in Example 1 except that the phenol concentration was 53% by volume in terms of the final concentration.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 3 in FIG. 4 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 240 ⁇ L of chloroform was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 4 in FIG. 4 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 4-bromoveratrole was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 1 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 6-bromo-1,4-benzodioxane was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 2 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 1-bromo-4-trifiuoromethoxybenzene was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 3 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 1-bromo-2,4-dimethoxybenzene was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 4 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 4-fluoroanisole was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 5 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of 4-bromotoluene was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 6 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • Example 2 The operation was carried out in the same manner as in Example 1 using serum as the biological sample, except that 100 ⁇ L of ethyl 4-bromobutyrate was added instead of 60 ⁇ L of p-bromoanisole to the homogenate.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 7 in FIG. 5 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity.
  • a solution was prepared by adding hydrochloric acid to the solution prepared in Example 1 such that the pH of the solution was adjusted to 4.2.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 1 in FIG. 6 .
  • Example 2 The same peak as in Example 1 was detected also in the case where the pH of the solution was 4.2, and it could be confirmed that only RNA was extracted with high purity.
  • a solution was prepared by adding hydrochloric acid to the solution prepared in Comparative Example 1 such that the pH of the solution was adjusted to 3.6.
  • Example 2 The operation was carried out in the same manner as in Example 1. The result is shown in lane 2 in FIG. 6 . The same three peaks as in Comparative Example 1 were observed. By this, it could be confirmed that, in cases where the solution containing 50% by volume of phenol is used, contamination with DNA fragments occurs also at a pH of 3.6.
  • Example 2 The operation was carried out in the same manner as in Example 1 except that cultured cells (HEK293 cells) suspended in 300 ⁇ L of PBS were used as the biological sample, instead of 300 ⁇ L of serum.
  • cultured cells HEK293 cells suspended in 300 ⁇ L of PBS were used as the biological sample, instead of 300 ⁇ L of serum.
  • Example 2 The operation was carried out in the same manner as in Example 1 except that 1.25 volumes, instead of 1.5 volumes, of ethanol was added to the aqueous layer.
  • Example 2 The operation was carried out in the same manner as in Example 1 except that 1.25 volumes, instead of 1.5 volumes, of ethanol was added to the aqueous layer.
  • Example 2 The operation was carried out in the same manner as in Example 1 except that “Agilent RNA 6000 nano kit” (model number, 5067-1511) (manufactured by Agilent Technologies Inc.) was used as the kit, instead of “Agilent RNA 6000 pico kit”. The results are shown in FIG. 7 .
  • the respective components of the solution were mixed such that their final concentrations were as described below, to prepare a solution for RNA extraction. That is, 60 ⁇ L of additional p-bromoanisole was added to 900 ⁇ L of the solution having the same composition as in Example 1, to prepare the solution.
  • the sample was homogenized.
  • the resulting homogenate was centrifuged at room temperature at 12,000 ⁇ G for 10 minutes. By this, an aqueous layer containing RNA, and an organic layer and an intermediate layer containing DNA and proteins were formed. From these, 350 ⁇ L of the aqueous layer was separated into another tube.
  • Example 2 The operation was carried out in the same manner as in Example 1. The results are shown in lanes 1 , 3 and 5 in FIG. 8 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity (lane 1 ). Since the peak in the RNase-treated sample (lane 3 ; RNase (+)) was as weak as that in BLANK (lane 5 ), it could be confirmed that the extracted nucleic acid contained only RNA.
  • the respective components of the solution were mixed such that their final concentrations were as described below, to prepare a solution for RNA extraction. That is, 90 ⁇ L of additional chloroform was added to 900 ⁇ L of the solution having the same composition as in Example 1, to prepare the solution.
  • the sample was homogenized.
  • the resulting homogenate was centrifuged at room temperature at 12,000 ⁇ G for 10 minutes. By this, an aqueous layer containing RNA, and an organic layer and an intermediate layer containing DNA and proteins were formed. From these, 350 ⁇ L of the aqueous layer was separated into another tube.
  • Example 2 The operation was carried out in the same manner as in Example 1. The results are shown in lanes 2 , 4 and 5 in FIG. 8 .
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity (lane 2 ). Since the peak in the RNase-treated sample (lane 4 ; RNase (+)) was as weak as that in BLANK (lane 5 ), it could be confirmed that the extracted nucleic acid contained only RNA.
  • the solution having the same composition as in Comparative Example 3 was prepared except that the phenol concentration was 55% by volume.
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity (lane 1 ). Since the peak in the RNase-treated sample (lane 2 ; RNase (+)) was as weak as that in BLANK (lane 5 ), it could be confirmed that the extracted nucleic acid contained only RNA.
  • Example 2 The same peak as in Example 1 was detected, and it could be confirmed that only RNA was extracted with high purity (lane 3 ). Since the peak in the RNase-treated sample (lane 4 ; RNase (+)) was as weak as that in BLANK (lane 5 ), it could be confirmed that the extracted nucleic acid contained only RNA.
  • Example 9 Composition of solution Phenol (% by volume) 58 55 65 53 58 58 58 58 Guanidinium thiocyanate (M) 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 Glycerol (% by volume) 5 5 5 5 5 5 5 5 5 Sodium acetate (M) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 2-Mercaptoethanol (% by — — — — — — — — — volume) pH of solution 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 .
  • Organic solvent (added later) Bromoan- Bromoan- Bromoan- Bromoan- Chloroform Bromovera- Benzo- Trifluoro- Dimethoxy isole isole isole isole isole trole

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CN108676791A (zh) * 2018-04-25 2018-10-19 浙江迪恩生物科技股份有限公司 一种磁珠法提取dna的试剂盒及提取方法
US10260063B2 (en) * 2014-10-24 2019-04-16 Abbott Molecular Inc. Enrichment of small nucleic acids
CN115386574A (zh) * 2022-09-02 2022-11-25 苏州英泽生物医药科技有限公司 一种不使用氯仿的核酸提取方法

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CN105431536B (zh) * 2013-08-16 2021-01-01 通用电气公司 用于提取和储存核酸的方法和组合物
CN103952399A (zh) * 2014-04-18 2014-07-30 天根生化科技（北京）有限公司 一种使用含有指示剂的酚胍盐裂解液提取核糖核酸的方法
SG11201608725YA (en) 2014-04-25 2016-11-29 Shire Human Genetic Therapies Methods for purification of messenger rna
KR20190014204A (ko) 2017-07-28 2019-02-12 가톨릭대학교 산학협력단 미세관을 이용한 rna 추출 방법
CN108587029B (zh) 2018-03-28 2020-09-08 中国石油天然气股份有限公司 一种相变材料液及其所形成的固相支撑剂
CN110887970B (zh) * 2019-11-29 2023-10-31 北京赛科希德科技股份有限公司 抽提缓冲液、兔脑抽提液、pt检测试剂及pt检测试剂盒
KR102587121B1 (ko) 2020-09-15 2023-10-11 주식회사 제노헬릭스 Rna 분리용 조성물
KR102657767B1 (ko) 2021-12-02 2024-04-16 한화오션 주식회사 액화가스 연료 선박의 시운전용 연료 공급 시스템
CN114350650A (zh) * 2021-12-16 2022-04-15 力因精准医疗产品（上海）有限公司 无dna残留血液rna提取试剂盒及核酸提取方法

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CN115386574A (zh) * 2022-09-02 2022-11-25 苏州英泽生物医药科技有限公司 一种不使用氯仿的核酸提取方法

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