JPH11196869A - Isolation of liponucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for isolating a liponucleic acid, capable of simply and safely isolating the liponucleic acid in high purity from a specimen such as cells, and to obtain a reagent therefor. SOLUTION: This method for isolating a liponucleic acid comprises mixing a specimen containing the liponucleic acid, an acidic solution containing a chaotropic agent, a water-soluble organic solvent and a nucleic acid-binding carrier to allow the carrier to adsorb the liponucleic acid, separating the carrier- liponucleic acid complex from the liquid phase, and, if necessary, subsequently washing the carrier-liponucleic acid complex to elute the liponucleic acid from the carrier-liponucleic acid complex.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for isolating a ribonucleic acid and a reagent therefor, and more particularly, to a method for easily and highly purifying a ribonucleic acid from a sample containing the ribonucleic acid using a nucleic acid-binding carrier. And a reagent therefor. The present invention can also be applied to an automatic nucleic acid extraction device.

[0002]

2. Description of the Related Art While deoxyribonucleic acid (DNA) plays a vital role in life information, ribonucleic acid (RNA) is a molecule that receives the information and plays an important role in vivo such as protein synthesis. Ribonucleic acid is largely divided into messenger RNA (mRNA) and transfer RNA (tRN).
A) and liposomal RNA (rRNA),
Each has different properties. Some viruses and the like use ribonucleic acids as information carriers. The analysis of these ribonucleic acids provides very important information in fields such as biochemistry, genetic engineering and clinical diagnosis, and the isolation of ribonucleic acids from biomaterials is an important and indispensable part of these analyses. Step. Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-
In order to obtain good results in analytical methods such as PCR), it is necessary to use ribonucleic acids with as high a purity as possible.

In general, it is necessary to disrupt cells in order to extract ribonucleic acid, and at this stage, the ribonucleic acid becomes a mixture with proteins, lipids, sugars, deoxyribonucleic acid, and the like. Since ribonucleic acid is easily decomposed by ribonuclease which is ubiquitous in the living body, its isolation is usually performed in a protein denaturing substance or an organic solvent capable of weakening the activity of ribonuclease. Among them, the most commonly used method is the so-called AGPC method [Analitycal Biochemistry 162, 156-159 (19)
87)], and (1) the biomaterial is dissolved in a guanidine thiocyanate solution, and an acidic buffer, a phenol solution,
A chloroform solution is sequentially added, and (2) the protein denatured with phenol and the insolubilized deoxyribonucleic acid are separated into an intermediate phase between an organic solvent phase and an aqueous phase by centrifugation. In this method, nucleic acids are insolubilized by adding isopropanol, and (4) only ribonucleic acids are selectively precipitated by centrifugation.
The AGPC method has an advantage that ribonucleic acid can be relatively easily and efficiently isolated as compared with a method for isolating ribonucleic acid using other ultracentrifugation methods. However, phenol or chloroform, which is a poisonous substance, must be used, and a relatively time-consuming step such as isopropanol precipitation is required. Requires a safer and simpler method.

On the other hand, as a simple method for extracting nucleic acids, a method using silica particles as a nucleic acid-binding carrier is described in Boom.
[J. Clin. Microbiol. 28 (3),
495-503 (1990)]. This method comprises the steps of (1) mixing a biomaterial, a neutral solution composed of guanidine thiocyanate, EDTA, and Triton X100, and a nucleic acid-binding solid phase (silica), binding the solid phase to a nucleic acid, and (2) The solid phase to which the nucleic acid is bound is separated from the adsorbent, (3) the solid phase is washed with a washing solution containing guanidine thiocyanate, (4) the solid phase is further washed with a 70% aqueous ethanol solution, 5) After washing with acetone, the solid phase is dried, and (6) the nucleic acid is eluted with a lysis solution. This method is characterized in that a nucleic acid is isolated from a sample such as a cell without using a highly toxic substance such as phenol and without using a concentration operation such as isopropanol precipitation. However, ribonucleic acid obtained from cells or the like by this method is not suitable for use in analyzing only ribonucleic acid because a large amount of deoxyribonucleic acid is mixed.

A method for isolating nucleic acids using a carrier such as silica particles is to bind nucleic acids in an agarose gel to the surface of glass particles in NaI (sodium iodide) and separate them [Proc. Natl. Acad. .Sci.USA, 76,615, (1979)]
Etc. have been reported. A common feature of these methods is that silica and nucleic acid are bound in a neutral solution containing chaotropic ions (anions having a large ionic radius) such as iodine ions and thiocyanate ions. These methods are mainly aimed at isolating deoxyribonucleic acid, and although separation of ribonucleic acid and the like is possible, yield is low and isolation of ribonucleic acid alone is not possible. Can be said to be unsuitable as an isolation method.

Further, a method of isolating ribonucleic acid (lithium precipitation method) utilizing the chemical property of insolubilizing ribonucleic acid by adding lithium ions to an aqueous solution of ribonucleic acid has been devised (Molecular Cloning, 1.4, (1989)). However, this method has problems such as the necessity of high-speed centrifugation as a step of precipitating ribonucleic acid. Thus, there is a need for a method that overcomes these problems.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method and a reagent for easily and safely isolating high-purity ribonucleic acid from a sample such as a cell.

[0008]

The present inventors have focused on a nucleic acid isolation method using a nucleic acid-binding carrier such as silica particles, and have conducted various studies. As a result, a chaotropic agent and a water-soluble organic solvent have been found. The yield and purity of isolated ribonucleic acid (ribonucleic acid selectivity) are dramatically improved by dissolving biological material such as cells using an acidic solution containing ribonucleic acid and contacting it with a nucleic acid binding carrier such as silica particles. To increase
The present invention has been completed.

That is, the present invention provides (1) mixing a sample containing a ribonucleic acid, an acidic solution containing a chaotropic agent, a water-soluble organic solvent and a nucleic acid-binding carrier, and allowing the ribonucleic acid to be adsorbed on the carrier; (2) separating the carrier-ribonucleic acid complex from the liquid phase, washing the carrier-ribonucleic acid complex if necessary, and (3) eluting the ribonucleic acid from the carrier-ribonucleic acid complex. This is a method for isolating ribonucleic acid.

The present invention is also a reagent for isolating ribonucleic acid, comprising an acidic solution containing a chaotropic agent, a water-soluble organic solvent and a nucleic acid-binding carrier, a washing solution and an eluate.

Furthermore, the present invention relates to a method for preparing a ribonucleic acid, a ribonuclease inhibitor, dN
This is a method for producing cDNA, comprising adding TPs, a reverse transcription primer, and a reverse transcription reaction buffer to cause a reaction to synthesize cDNA from ribonucleic acid.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The sample containing ribonucleic acid according to the present invention includes, for example, serum, blood, cerebrospinal fluid, tissue, urine, feces,
Cells and cultured cells separated from biological materials such as saliva, semen, and blood. The ribonucleic acid may also contain exogenous ribonucleic acids such as viruses, bacteria, and fungi, or ribonucleic acids synthesized enzymatically in vitro, in addition to endogenous ribonucleic acids derived from these samples.

In the present invention, first, a ribonucleic acid-containing sample, an acidic solution containing a lithium salt and a chaotropic agent, a water-soluble organic solvent, and a nucleic acid-binding carrier are mixed, and the ribonucleic acid is adsorbed on the carrier. . The acidic solution and the water-soluble organic solvent containing the lithium salt and the chaotropic agent of the present invention are a solution for dissolving and adsorbing ribonucleic acid.

The lithium salt used in the present invention includes an inorganic lithium salt and an organic lithium salt. Examples thereof include lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide or lithium borate, lithium acetate, and citric acid. There is no particular limitation as long as lithium ions can be generated in an aqueous solution of lithium or the like, and lithium chloride is particularly preferred. The concentration of the lithium salt is preferably used in the range of 0.1M to 2M. Lithium salts are known to more easily coordinate to ribonucleic acids than monovalent cations having a large ionic radius, for example, other salts such as sodium salts.

Conventionally, a method for precipitating high molecular weight ribonucleic acid using lithium ions has been widely used [Mo
lecular Cloning, 1.4, (1989)]. However, these methods are characterized in that they are used for insolubilizing ribonucleic acid in a solution, and the insolubilized ribonucleic acid in the solution is recovered by precipitation or filtration by centrifugation. On the contrary, in the present invention, it is important to adsorb ribonucleic acid to a nucleic acid binding solid phase such as silica under conditions that do not insolubilize ribonucleic acid.

In this application, it is effective to include a protein denaturing substance such as a guanidine salt and urea in the reaction solution. In fact, according to the method of the present invention, no insolubilized component is observed when an appropriate amount of sample is used. However, when the number of samples is extremely large, a phenomenon in which the insolubilized components aggregate may be observed. Therefore,
In the present invention, when ribonucleic acid and other components are insolubilized, it is desirable to remove them or reduce the amount of the sample.

By chaotropic agent is meant a substance capable of changing the secondary, tertiary or quaternary structure without affecting the primary structure of proteins and nucleic acids. The chaotropic agent used in the present invention includes a compound selected from the group consisting of a guanidinium salt, urea, iodide, perchlorate and (iso) thiocyanate. Although the concentration of the chaotropic agent varies depending on each compound, it is usually 1 to 20M.
It is.

The guanidine salts used in the present invention include guanidine inorganic salts and guanidine organic salts. The guanidine salt is not particularly limited as long as it is a guanidine salt generally used for denaturing proteins, such as guanidinium hydrochloride, guanidinium acetate, guanidinium phosphate, guanidinium (iso) thiocyanate, guanidinium sulfate or guanidinium carbonate. They may be used in combination, and the guanidine salt is preferably used at a high concentration of 5 M or more.

The iodide used in the present invention includes sodium iodide or potassium iodide, and the perchlorate includes sodium perchlorate, potassium perchlorate, lithium perchlorate or ammonium perchlorate. And the like, and as the (iso) thiocyanate,
Examples include sodium (iso) thiocyanate, potassium (iso) thiocyanate, and ammonium (iso) thiocyanate.

The water-soluble organic solvent used in the present invention includes isopropanol, propanol and ethanol. Particularly preferred is ethanol. The concentration of the water-soluble organic solvent is about 1-99%, preferably about 1-50%, and more preferably about 5-20%. The water-soluble organic solvent may be added to the acidic solution as the dissolution adsorption solution, or may be added separately. In the absence of a water-soluble organic solvent, the isolation yield of ribonucleic acid is reduced by 30-60%.

Further, the dissolving / adsorbing solution may contain a surfactant for the purpose of breaking down cell membranes or solubilizing proteins contained in cells. As a surfactant,
Although it is not particularly limited as long as it is generally used for extracting nucleic acids from cells or the like, specifically, nonionic surfactants such as Triton surfactants and Tween surfactants, N-lauroyl Anionic surfactants such as sodium sarcosinate can be mentioned. In the present invention, it is preferable to use 0.01 to 0.5% of a nonionic surfactant.

Some types of samples cannot be dissolved by the dissolution / adsorption solution of the present invention. For example, plants, yeast,
Molds and certain gram-positive bacteria, for example, have special cell wall structures and it may be difficult to directly isolate ribonucleic acids using the methods of the present invention. When isolating ribonucleic acid from such a material, it is necessary to perform a pretreatment suitable for each sample, and the sample after the pretreatment may be treated using the method of the present invention.

In the present invention, a sample dissolved in the above-mentioned dissolving and adsorbing solution is brought into contact with a nucleic acid-binding carrier such as silica particles under acidic conditions of pH 6.0 or less. For this purpose, the lysis adsorbent must be buffered with a suitable buffer. At this time, the buffer used is p
There is no particular limitation as long as H is 6.0 or less. In the present invention, the use of an acetate buffer or a citrate buffer having a pH of 3 to 4 is most preferred.

The present invention is characterized by using a nucleic acid-binding carrier capable of binding to ribonucleic acid in the above-mentioned dissolved and adsorbed solution, for example, silica particles. The term silica as used herein also includes silicon dioxide crystals and other silicon oxides, diatomaceous earth, glass powder, chemically modified silica, and composites of silica with ultra-normal metal oxides. Also, use of cellulose, nitrocellulose, latex, hydroxyapatite, etc. can be easily considered. Specific examples of the form of the nucleic acid binding carrier include, but are not limited to, particles, filters, and reaction vessels. Of these,
In consideration of the efficiency of adsorption and elution, the form of the particles is more preferable, and in view of the efficiency of adsorption and elution, in the present invention, a particle size of 0.05 to 500 μm is more preferable.
These particle sizes are preferably selected according to the respective applications. The used amount of the carrier is 1 to 10%, and the used amount varies depending on the type of the sample.

In the present invention, the step of separating the carrier-ribonucleic acid complex obtained in the above step from the liquid phase includes, for example, centrifugation, removal of the supernatant, or the use of a magnetic field to separate the carrier-ribonucleic acid from the liquid phase. This is a step of separating the complex. In the case of a filter, a reaction vessel, or the like, it is only necessary to discharge or remove the liquid.

In the present invention, the step of washing the carrier-ribonucleic acid complex obtained in the above step is to suspend the complex with an appropriate washing solution using, for example, a vortex mixer, and re-suspend the liquid phase. This is a step of further separating and removing the supernatant. Separation of the complex is preferably performed by centrifugation, filtration, column operation, or the like. Further, if a nucleic acid binding carrier containing a superparamagnetic metal oxide in the particles is used, a simple magnetic separation method using a magnet or the like can be used. Is possible, which is more preferable.

It is preferable to use a chaotropic agent, particularly guanidine hydrochloride, as the washing solution in the present invention.
The concentration of the guanidine salt in the washing solution is preferably 6M or more. The solution may contain a surfactant and a water-soluble organic solvent, and the pH is not particularly limited.

In the present invention, the carrier-ribonucleic acid complex obtained by the step of washing with a washing solution containing a chaotropic agent is preferably further washed with a low salt buffer. The term "low salt concentration" as used herein means a salt concentration that does not extremely inhibit the reverse transcription reaction or the like when this buffer is mixed into the final ribonucleic acid eluate, and includes simple water. In the present invention, a buffer of 100 mM or less is preferable. The buffer is preferably a Tris buffer, but is not particularly limited. Further, this solution may contain a surfactant, and the pH is not particularly limited.

In a conventional method for separating nucleic acids using a carrier,
Since an organic solvent such as ethanol or acetone is used in this washing step, the carrier must be dried. In the present invention, the ribonucleic acid in the next step can be eluted without a drying step. This is very useful in shortening the isolation time of ribonucleic acid, and is most preferable in preventing contamination in an open system during drying of particles. The term “contamination” as used herein refers to contamination between samples and contamination of amplified nucleic acid generated by polymerase chain reaction (PCR) or the like. These contaminations are considered to be the biggest causes of erroneous judgment in analysis of infectious diseases and the like using RT-PCR and the like.

The ribonucleic acid elution step in the present invention is a step of eluting the ribonucleic acid from the nucleic acid-binding carrier to which the ribonucleic acid has been adsorbed. As an eluent used for this purpose, any one that promotes elution of ribonucleic acid from a carrier may be used.
There is no particular limitation. Specifically, water or Tris-E
DTA buffer [10 mM Tris buffer, 1 mM EDT
A, pH 8.0] is preferred.

The elution may be promoted by heating. The heating temperature is not particularly limited as long as it does not adversely affect the ribonucleic acid, but in the present invention, it is preferably around 60 ° C. The ribonucleic acid eluted in this way does not undergo desalting and concentration operations such as dialysis and ethanol precipitation,
It can be directly used in an enzyme reaction using a reverse transcriptase or the like. Further, the carrier-ribonucleic acid complex can be directly subjected to an enzymatic reaction without being eluted from the solid phase.

The method for isolating ribonucleic acid according to the present invention enables ribonucleic acid with little contamination with deoxyribonucleic acid or the like to be separated from biological components at high efficiency by a simple operation without using dangerous solvents and the like. It is clear that the present invention can be applied to a ribonucleic acid purification kit and a nucleic acid extraction apparatus in which solid phase separation operation and reagent dispensing operation are automated. Further, the ribonucleic acid obtained by the method of the present invention was analyzed by Northern blotting analysis, RT
-It can be used as a template for amplification by PCR analysis and the so-called NASBA method described in European Patent EP 0 329 822.

One embodiment of the present invention comprises the steps of (1) mixing a ribonucleic acid-containing sample, an acid solution containing a lithium salt and a chaotropic agent, a water-soluble organic solvent, and a nucleic acid-binding carrier to mix ribonucleic acid. Adsorbed on the carrier, (2) carrier-
The ribonucleic acid complex is separated from the liquid phase and, if necessary,
A method for isolating a ribonucleic acid, comprising washing the ribonucleic acid complex and (3) eluting the ribonucleic acid from the carrier-ribonucleic acid complex.

In another embodiment of the present invention, there are provided (1) a sample containing ribonucleic acid, an acidic solution containing 0.1 to 2 M lithium salt and 3 to 7 M guanidinium salt or urea, 5 to 20 M % Of isopropanol, propanol and ethanol, and a nucleic acid-binding carrier, and a ribonucleic acid is adsorbed to the carrier, and (2) the carrier-ribonucleic acid complex is separated from the liquid phase. Separation, washing the carrier-ribonucleic acid complex if necessary, (3)
A method for isolating ribonucleic acid, comprising eluting ribonucleic acid from the carrier-ribonucleic acid complex.

Further, another embodiment of the present invention relates to (1) a sample containing ribonucleic acid, lithium chloride, lithium acetate,
One or more lithium compounds selected from the group consisting of lithium citrate, lithium carbonate, lithium hydroxide and lithium borate, and guanidinium salts, ureas, iodides, perchlorates and (iso) thiocyanates An acidic solution containing at least one compound selected from the group consisting of: pH 6.0 or less, a water-soluble organic solvent selected from the group consisting of isopropanol, propanol and ethanol, and a superparamagnetic metal oxide (2) using a magnetic field to separate the carrier-ribonucleic acid complex from the liquid phase, and (3) further transforming the carrier-ribonucleic acid complex into guanidinium. Washing with a washing solution containing a compound selected from the group consisting of salt, urea, iodide, perchlorate and (iso) thiocyanate Using a magnetic field, the carrier-ribonucleic acid complex is separated from the liquid phase. (4) Subsequently, the carrier-ribonucleic acid complex is washed with a low salt concentration buffer of 100 mM or less, and the carrier-ribonucleic acid complex is separated from the liquid phase using a magnetic field. (5) A method for isolating ribonucleic acid, further comprising eluting the ribonucleic acid from the carrier with a solution for eluting ribonucleic acid.

In another embodiment of the present invention, there is provided (a) one or more kinds selected from the group consisting of lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide and lithium borate. PH 6 containing a lithium compound and one or more compounds selected from the group consisting of compounds selected from the group consisting of guanidinium salts, urea, iodide, perchlorates and (iso) thiocyanates A dissolving adsorbent comprising an acidic solution of 0.0 or less, and a water-soluble organic solvent selected from the group consisting of isopropanol, propanol and ethanol;
(b) a silica carrier containing a superparamagnetic metal oxide, (c) a washing solution containing a compound selected from the group consisting of guanidinium salts, ureas, iodides, perchlorates and (iso) thiocyanates, (d) ) A reagent for ribonucleic acid isolation comprising a washing solution that is a low salt buffer of 100 mM or less and (e) a solution for eluting ribonucleic acid from a carrier.

Another embodiment of the present invention relates to (a) 0.1 to 2
M lithium chloride, lithium acetate, lithium citrate,
One or more compounds selected from the group consisting of lithium carbonate, lithium hydroxide and lithium borate;
3 to 7M containing one or more compounds selected from the group consisting of guanidinium salts, ureas, iodides, perchlorates and (iso) thiocyanates. A ribonucleic acid dissolution and adsorption solution containing an acidic solution having a pH of 6.0 or less and a water-soluble organic solvent selected from the group consisting of 5 to 20% of isopropanol, propanol and ethanol; (b) containing a superparamagnetic metal oxide (C) a washing solution containing a compound selected from the group consisting of a guanidinium salt, urea, iodide, perchlorate and (iso) thiocyanate, and (d) a buffer solution having a low salt concentration of 100 mM or less. This is a reagent for isolating ribonucleic acid, including a washing solution and (e) a solution for eluting ribonucleic acid from a carrier.

The method for producing the cDNA of the present invention comprises the steps of: adding a ribonucleic acid, a ribonuclease inhibitor, dNTPs, a reverse transcription primer, The reaction is performed by adding a buffer for reverse transcription reaction, and cDNA is synthesized from ribonucleic acid. As the reverse transcriptase, AMV reverse transcriptase, M-MLV reverse transcriptase, Tth DNA polymerase, or the like is used. As the ribonuclease inhibitor, a human placenta-derived ribonuclease inhibitor or the like is used. dNTPs are dATP, dCT
It is a mixture of P, dGTP and dTTP. As a reverse transcription primer, a sequence-specific primer, an oligo dT primer, a random primer, or the like is used. As the buffer for the reverse transcription reaction, a buffer containing an inorganic salt such as Mg or Mn and KCl, the pH of which is adjusted so as to optimize the reverse transcription reaction, is used.

[0039]

The effects of the present invention will be further clarified by exemplifying embodiments of the present invention.

Example 1 Extraction of ribonucleic acid from yeast (1) Preparation of yeast cells cerevisiae (ATCC236
6) using a YPD medium [1% yeast extract, 1% tryptone,
1% D-(+)-glucose anhydrous solution (pH 5.3)] at 30 ° C for 16 hours. Thereafter, the culture solution was separated by centrifugation (5,000 rpm × 3 minutes), and the supernatant was removed to collect the bacteria. Thymolase solution (3
% Thymolase, 0.9M sorbitol, 0.05M D
After TT) was added and completely suspended, 37 ° C., 10
The cell wall was broken by reacting for minutes. After centrifugation, the pellet obtained by removing the supernatant was used as an RNA extraction material.

(2) Extraction of ribonucleic acid In a microtube, 700 μl of the dissolved adsorption solution [6 M guanidine hydrochloride, 1 M lithium chloride, 0.2 M sodium acetate— was added to one platinum loop of the yeast pellet prepared in (1). Hydrochloric acid buffer (pH 3.0), 0.1% Triton X10
0, 1.4M 2-mercaptoethanol, and 0.1
0, 20, 30, or 40% (v / v) ethanol] to completely lyse the cells. Magnetic silica particles (particle size: 1 to 10 μm, containing 30% of iron trioxide particles, specific surface area: 280 m ² / g, surface pore diameter: 2 to 6 nm, pore volume: 0.025 ml) / G: manufactured by Suzuki Yushi Co., Ltd.), and mixed with a vortex mixer at room temperature for 10 minutes. Next, place the microtube on a magnetic stand (MCP-M: manufactured by Dinal Corporation).
The magnetic silica particles were collected, and the supernatant was removed with a pipette. Next, the microtube was removed from the magnetic stand, and 0.6 ml of the washing solution [6 M guanidine hydrochloride, 0.2 M sodium acetate-hydrochloric acid buffer (pH 4.
0)] and add about 10 with a vortex mixer.
After stirring for 2 seconds, the microtube was placed on a magnetic stand again, the magnetic silica particles were collected, and the supernatant was removed with a pipette. Further, after performing a washing operation twice with 0.8 ml of 10 mM Tris buffer (pH 6.4), the buffer was completely removed, and Tris-EDTA buffer (10 mM Tris buffer, 1 mM EDTA (pH 8. 0)) 50 μl
Was added, and the magnetic silica particles were suspended by pipetting. Thereafter, the mixture was heated at 65 ° C. for 2 minutes, again set on a magnetic stand, magnetic silica was collected, and the supernatant was recovered.

(3) Analysis of ribonucleic acid by agarose electrophoresis 9 μm of ribonucleic acid solution obtained from yeast by the method of the present invention
and 1 μl of a dye solution (50% glycerol, 0.25% bromophenol blue) were mixed and provided to a slot of a 1% agaroloth gel. The electrophoresis apparatus used was Mupid (manufactured by Cosmo Bio Inc.) and used a 1 × TBE buffer (89 mM).
Tris-HCl, 89 mM boric acid, 2.5 mM EDTA
Electrophoresis was performed at 100 V for 30 minutes in 2Na). After completion of the electrophoresis, the gel was immersed in an ethidium bromide solution for 30 minutes, washed lightly with tap water, and photographed under a UV irradiation using a polaroid camera for fluorescence of nucleic acids.

In this method, the ribonucleic acid 28S r
RNA, 18S rRNA and tRNA could be detected. The result is shown in FIG. In FIG. 1, lane 1
Is a molecular weight marker composed of HindIII digest of lambda phage DNA, lane 2 is 0% ethanol concentration in the dissolved adsorbent, lane 3 is 10% ethanol in the dissolved adsorbent, and lane 4 is ethanol concentration in the dissolved adsorbent 20
%, Lane 5 is 30% ethanol concentration in the dissolved adsorbent,
Lane 6 shows the migration pattern of the ribonucleic acid extracted and isolated at an ethanol concentration of 40% in the dissolved adsorbent. As is clear from FIG. 1, the ethanol concentrations are 0%, 10%, and 20%.
RNA recovery is high. Table 1 shows the results of accurately measuring the amount of recovered RNA with an absorptiometer.

[0044]

[Table 1]

As shown in Table 1, an ethanol concentration of 10
%, Especially when 10% is added among 20%, RNA
The recovery amount was the largest. TRN by adding ethanol
This is probably due to an increase in the recovery of small RNAs such as A.

Example 2 Extraction of ribonucleic acid from plant tissue (1) Pretreatment of tobacco green leaf tissue After collecting tobacco green leaf, liquid nitrogen was immediately added and flash frozen. The frozen tissue (1-10 g) was placed in a mortar and crushed with a pestle. During the crushing, liquid nitrogen was poured before the plant tissue was melted, and care was taken not to melt the plant tissue.
The process was performed until most of the tissues became fine particles of 0.5 mm or less.

(2) Extraction of ribonucleic acid In a microtube, 700 μl of the dissolved adsorbent [6 M guanidine hydrochloride, 1 M lithium chloride, 0.2 M sodium acetate, was added to 0.1 g of the powder tissue prepared in (1).
Hydrochloric acid buffer (pH 3.0), 0.1% Triton X1
00, 1.4 M 2-mercaptoethanol and 0,
5, 10, 15, 20% (v / v) ethanol] to completely lyse the cells. Magnetic silica particles (particle size: 1 to 10 μm, containing 30% of iron trioxide particles, specific surface area: 280 m ² / g, surface pore diameter: 2 to 6 nm, pore volume: 0.025 ml) / G: manufactured by Suzuki Yushi Co., Ltd.), and mixed with a vortex mixer at room temperature for 10 minutes. Next, the microtube was set on a magnetic stand (MCP-M: manufactured by Dinal Corporation) to collect the magnetic silica particles, and the supernatant was removed with a pipette. Next, the microtube was removed from the magnetic stand, and 0.6 ml of the washing solution [6 M guanidine hydrochloride, 0.2 M sodium acetate-hydrochloric acid buffer (pH 4.
0)] and add about 10 with a vortex mixer.
After stirring for 2 seconds, the microtube was placed on a magnetic stand again, the magnetic silica particles were collected, and the supernatant was removed with a pipette. This operation is repeated four times. In addition, 0.8m
After washing twice with 1 l of 10 mM Tris buffer (pH 6.4), the buffer was completely removed, and Tris-ED was removed.
TA buffer (10 mM Tris buffer, 1 mM EDTA
(PH 8.0)), and the magnetic silica particles were suspended by pipetting. Thereafter, the mixture was heated at 65 ° C. for 2 minutes, again set on a magnetic stand, magnetic silica was collected, and the supernatant was recovered.

(3) Analysis of ribonucleic acid by agarose electrophoresis 9 μl of ribonucleic acid solution obtained from tobacco green leaf tissue by the method of the present invention and a dye solution (50% glycerol, 0.25
% Bromophenol blue) and mixed into a 1% agaroloth gel slot. The electrophoresis device is Mu
1 × TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDT) using pid (manufactured by Cosmo Bio).
A. 2Na) was run at 100 V for 30 minutes. After the electrophoresis is completed, the gel is placed in an ethidium bromide solution for 30 minutes.
After immersion for 5 minutes and washing lightly with tap water, the fluorescence of the nucleic acid was photographed using a Polaroid camera under UV irradiation.

In this method, nuclear-derived ribonucleic acids (28S rRNA and 18S rRNA) and chloroplast-derived ribonucleic acids (23S rRNA and 16S rR) are used.
NA) and tRNA could be detected. The result is shown in FIG. In FIG. 2, lane 1 is a molecular weight marker consisting of a HindIII digest of lambda phage DNA,
Lanes 2 and 3 have 0% ethanol concentration in the dissolved adsorbent, lanes 4 and 5 have 5% ethanol concentration in the dissolved adsorbent, lanes 6 and 7 have 10% ethanol concentration in the dissolved adsorbent, and lanes 8 and 9 have Lanes 10 and 11 show the migration pattern of ribonucleic acid extracted and isolated at the ethanol concentration of 20% in the dissolved and adsorbed solution. FIG.
As is clear from the above, in the extraction of RNA from tobacco green leaf tissue, the amount of RNA recovered increased by the addition of ethanol, and was best when the ethanol concentration was 10 to 20%.

[0050]

According to the present invention, high-purity ribonucleic acid can be easily, rapidly and safely isolated from various biological materials. The ribonucleic acid obtained by this method can be applied to various analyzes such as Northern blot analysis and RT-PCR analysis. By using this method, compared to the conventional method,
Time can be significantly reduced and reproducible and reliable results are obtained.

[Brief description of the drawings]

FIG. 1 is a photograph instead of a drawing, showing the results of electrophoretic analysis of ribonucleic acid isolated from yeast by the method of the present invention.

FIG. 2 is a photograph replacing a drawing, showing the results of electrophoretic analysis of ribonucleic acid isolated from tobacco green leaf tissue by the method of the present invention.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Fumiyoshi Kawakami 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyobo Co., Ltd. Inside Tsuruga Bio-Research Laboratory (72) Inventor Yoshihisa Kawamura 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyobo Co., Ltd., Tsuruga Bio Research Laboratory

Claims (10)

[Claims] (1) mixing a sample containing ribonucleic acid, an acidic solution containing a chaotropic agent, a water-soluble organic solvent, and a nucleic acid-binding carrier, and adsorbing the ribonucleic acid to the carrier; Separating the carrier-ribonucleic acid complex from the liquid phase, washing the carrier-ribonucleic acid complex if necessary, and (3) eluted the ribonucleic acid from the carrier-ribonucleic acid complex Isolation method. 2. (1) A sample containing ribonucleic acid, an acidic solution containing a lithium salt and a chaotropic agent, a water-soluble organic solvent and a nucleic acid-binding carrier are mixed, and the ribonucleic acid is adsorbed on the carrier. (2) separating the carrier-ribonucleic acid complex from the liquid phase, washing the carrier-ribonucleic acid complex if necessary, and (3) eluting the ribonucleic acid from the carrier-ribonucleic acid complex. For isolating ribonucleic acids. 3. The method according to claim 1, wherein the water-soluble organic solvent is isopropanol,
The method for isolating ribonucleic acid according to claim 1 or 2, which is a solvent selected from the group consisting of propanol and ethanol. 4. The method according to claim 1, wherein the water-soluble organic solvent is about 5 to 20% ethanol. 5. The single ribonucleic acid according to claim 1, wherein the chaotropic agent is a compound selected from the group consisting of guanidinium salts, urea, iodides, perchlorates and (iso) thiocyanates. Release method. (1) a sample containing ribonucleic acid, 0.1 to
Mixing an acidic solution containing 2M lithium salt and 3-7M guanidinium salt or urea, 5-20% of a water-soluble organic solvent selected from the group consisting of isopropanol, propanol and ethanol, and a nucleic acid binding carrier, The ribonucleic acid is adsorbed on the carrier, (2) the carrier-ribonucleic acid complex is separated from the liquid phase, and if necessary, the carrier-ribonucleic acid complex is washed. A method for isolating a ribonucleic acid, which is eluted from a complex. 7. A sample containing ribonucleic acid, one or more lithium selected from the group consisting of lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide and lithium borate. A compound and one selected from the group consisting of guanidinium salts, ureas, iodides, perchlorates and (iso) thiocyanates
Mixing an acidic solution having a pH of 6.0 or less containing one or more compounds, a water-soluble organic solvent selected from the group consisting of isopropanol, propanol and ethanol and a silica carrier containing a superparamagnetic metal oxide,
The ribonucleic acid is adsorbed on the carrier, (2) the carrier-ribonucleic acid complex is separated from the liquid phase using a magnetic field, and (3) the carrier-ribonucleic acid complex is further separated.
The ribonucleic acid complex is converted to guanidinium salt, urea, iodide,
After washing with a washing solution containing a compound selected from the group consisting of perchlorate and (iso) thiocyanate, the carrier-ribonucleic acid complex is separated from the liquid phase using a magnetic field. Washing with the following low salt concentration buffer, the carrier-ribonucleic acid complex was separated from the liquid phase using a magnetic field, (5)
A method for isolating ribonucleic acid, further comprising eluting the ribonucleic acid from the carrier with a solution for eluting ribonucleic acid. 8. A reagent for isolating ribonucleic acid, comprising an acidic solution containing a chaotropic agent, a water-soluble organic solvent and a nucleic acid-binding carrier, a washing solution, and an eluate. 9. (a) one or more lithium compounds selected from the group consisting of lithium chloride, lithium acetate, lithium citrate, lithium carbonate, lithium hydroxide and lithium borate, and a guanidinium salt, urea,
An acidic solution having a pH of 6.0 or less containing one or more compounds selected from the group consisting of iodides, perchlorates and (iso) thiocyanates, and isopropanol , A dissolved adsorption solution containing a water-soluble organic solvent selected from the group consisting of propanol and ethanol, (b) a silica carrier containing a superparamagnetic metal oxide, (c) a guanidinium salt, urea, iodide, perchlorate And a washing solution containing a compound selected from the group consisting of (iso) thiocyanate, (d) 100 mM
A reagent for isolating ribonucleic acid, comprising: a washing solution that is a buffer having a low salt concentration described below; 10. A reverse transcriptase, a ribonuclease, a ribonucleic acid isolated by the method according to claim 1, 2, 6 or 7, or a carrier-ribonucleic acid complex in the method according to claim 1, 2, 6 or 7. A method for producing cDNA, comprising adding an inhibitor, dNTPs, a primer for reverse transcription, and a buffer for a reverse transcription reaction and reacting to synthesize cDNA from ribonucleic acid.