CN106065400A - A kind of ribonucleic acid protective agent, test kit, application and store method - Google Patents

Abstract

The present invention provides a kind of ribonucleic acid protective agent and contains this protectant test kit and application and the store method of ribonucleic acid, and described protective agent comprises the aqueous trehalose that concentration is 0.27M-1M.Using the protective agent of the present invention, the holding time is long, to temperature-insensitive, uses temperature wide, and the extracellular rna sample after this protective agent processes does not affects such as cDNA synthesis, and the enzymatic activity in storehouse is built in PCR reaction and order-checking.Problem, the particularly preservation to extracellular Microrna, Reusability and the long-distance transports such as RNA degraded that this protective agent can efficiently solve to run into when exRNA preserves for a long time or uses and pollution are significant.

Description

A kind of ribonucleic acid protective agent, test kit, application and store method

Technical field

The present invention relates to biology field, particularly to a kind of RNA protective agent and test kit, application and store method.

Background technology

Ribonucleic acid (is abbreviated as RNA, i.e. Ribonucleic Acid), it is present in the carrier of genetic information in biological cell and fractionated viral, viroid, RNA is the long chain molecule being condensed through phosphide key by ribonucleotide, every vital movement of cell can be participated in, be the object of primary study in current medical science, biology and pharmaceutical field.

Along with the further investigation of RNA, find to have vital movement cell can from intracellular secretory to blood circulation or in specific tissue microenvironment, this kind of RNA be defined as extracellular rna (exRNA) by specific RNA.Owing to the RNA that exRNA is common with cell has obvious diversity, therefore exRNA can play various potential biological actions as signal.Medical science and pharmaceutical field increasingly pay attention to the target checked order by extracellular rna as potential drug and the degree of depth at present, for diagnosis and the targeted therapy of tumor, the purity of the Microrna in particularly exRNA and the active target spot specificity for targeted drug, there is significant impact.And exRNA long-term preserving, freeze thawing and the most often being degraded by RNase, what this was exRNA be widely used, store and transport brings great difficulty.

In the actual application of RNA, the preservation of RNA depends on and does not contains the pure water of RNase or add the aqueous solution of the chelating agen such as EDTA to dissolve RNA and can preserve in the ultra cold storage freezer of-80 DEG C 1 year, and for the RNA of sample size pettiness, irreversible RNA can be caused during the most frozen to-80 DEG C to rupture, Microrna biological activity originally can be destroyed at-80 DEG C of physical damage recovered to room temperature condition again equally.

There is some commercialization reagent at present on the market for preserving the biological specimen containing RNA, RNA later such as LifeTech company, the RNA stable etc. of Biomatrica company, either preserve with liquid or solid-state, enzyme in these reagent reaction suppressor alive and denaturant can protect RNA sample not degraded by RNase, but the defect of these reagent is used further to subsequent bio reaction after being to need to be purified original RNA sample, and purge process can further result in the loss of exRNA sample, and the solid-state of exRNA Microrna the most therein after purification preserves significant for long-distance transport and the Reusability of Microrna.

In sum, how ensure that exRNA sample after purification is protected under non-super low temperature condition, it is ensured that Microrna sample after purification still possesses high-quality and purity, how in long-distance transportation, still there is efficient biological activity it is critical that.

Therefore; this area can efficiently suppress RNase to degrade in the urgent need to exploitation one; avoid the RNA damage that multigelation causes, the most do not affect the various enzyme of follow-up exRNA and live the protective agent of reaction, this for current medical science, pharmacy and in biology the various researchs of exRNA be very important.

Summary of the invention

One of goal of the invention of the present invention, is the problem for above-mentioned existence, it is provided that even if a kind of lyophilizing repeatedly still ensures that quality and the purity of exRNA, and do not affect the reaction alive of follow-up various enzymes and the bioactive protective agent of exRNA.

First aspect present invention, it is provided that a kind of ribonucleic acid protective agent, described ribonucleic acid protective agent is for comprising:

Molar concentration is the trehalose of 0.27-1M,

Remaining is pure water.

Described pure water does not comprise ribonuclease.

It is also preferred that the left above-mentioned trehalose concentration is 0.27-0.5M.

In a particular embodiment, above-mentioned protective agent also comprises ribonuclease inhibitor, and described ribonuclease inhibitor is 1:(2000-20000 with the volume ratio of aqueous trehalose).

It is also preferred that the left also comprise ethylenediaminetetraacetic acid in above-mentioned protective agent, the content of described ethylenediaminetetraacetic acid is 0.2mM-0.5mM.In a particular embodiment, above-mentioned protective agent also comprises buffer, after described buffer adds protective agent, make protectant pH value range between 5.2 8.0.

Above-mentioned buffer is TE buffer, and the content of ethylenediaminetetraacetic acid is 0.2mM-0.5mM.

More preferably, above-mentioned trehalose concentration is 0.5M.

Second aspect present invention, it is provided that a kind of ribonucleic acid protective agent test kit, described test kit includes above-mentioned ribonucleic acid protective agent.

A third aspect of the present invention, it is provided that above-mentioned ribonucleic acid protective agent is preserving containing the application in the rna sample of any fragment length.

It is also preferred that the left above-mentioned ribonucleic acid is vitro rna.

A fourth aspect of the present invention, it is provided that the store method of a kind of ribonucleic acid, described method includes step:

A) vitro rna sample before purification or after purification is dissolved in above-mentioned ribonucleic acid protective agent；

B) solution that step a) obtains is made lyophilized powder；

C) lyophilized powder that step b) obtains is saved in room temperature and temperature below.

Above-mentioned storage temperature is less than 25 DEG C.

It is also preferred that the left above-mentioned storage temperature is less than-20 DEG C.

Above-mentioned ribonucleic acid is vitro rna.

Trehalose is also known as Radix Rhapontici sugar, gill fungus sugar etc..It it is a kind of safe and reliable natural saccharide.Trehalose is with 1 by two glucose molecules, the nonreducing sugar that 1-glycosidic bond is constituted, and is all widely present in nature in many edible animals and plants and microbial body.Trehalose can form the protecting film of uniqueness under the severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration at cell surface, and protected protein matter molecule invariance inactivates effectively, thus the life process of the body that sustains life and biological characteristic.A lot of application has been had about trehalose.Present inventor passes through great many of experiments, obtains above-mentioned technical scheme.Trehalose belongs to chemically inert material, has high stability after being dissolved in pure water under the conditions of room temperature (less than 25 DEG C) or 2 DEG C-8 DEG C.The trehalose of 0.27M-0.5M can preserve (24 months internal stabilities and chemical property are constant) in above-mentioned environment for a long time.And the trehalose of this concentration range is it can be avoided that the survival of the microorganisms such as antibacterial; there is the growth significantly suppressing the microorganisms such as bacterial fungus; this is necessary for protection Microrna; otherwise the metabolism of the microorganism such as bacterial fungus can produce RNase, is unfavorable for preservation and the Reusability of Microrna.

In this application, the aqueous trehalose that we are more than 0.27M concentration calls high concentration aqueous trehalose.Solution concentration unit " M " in the present invention is " mol/L ", i.e. molar concentration.

It is demonstrated experimentally that the dry powder that the aqueous trehalose of 0.27M-1M is formed after mixing with exRNA can preserve 18 months under the conditions of-20 DEG C, preserve under the conditions of 2 DEG C-8 DEG C 6 months, preserve 1 month at ambient temperature, be therefore easy to long-distance transportation or transnational mailing.

The trehalose of 0.27M-0.5M is sufficient for protecting exRNA to be because it can form close hydrogen bond with the phosphate group of ribonucleic acid, thus avoid exRNA to be exposed to RNase, the phosphate group concurrently formed is mutually exclusive, therefore the aqueous trehalose in this concentration range has RNA dissolubility more higher than pure water, under same solvent volume, aqueous trehalose can dissolve more exRNA, in room temperature, after under the conditions of 2 DEG C-8 DEG C and-20 DEG C, solid-state preserves and again dissolves, the special sign of RNA is compared through PAGE electrophoresis, 28s, 18s and 5s band is all without degraded conditions of streaking.

The RNA later of LifeTech company ensure that the RNA under dissolution conditions is not degraded by RNase, but but can bring RNA fracture equivalent damage through repeatedly multigelation, the RNA stable of Biomatrica company is also that RNA can be lyophilized into solid-state, but through repeatedly dissolving the RNase degraded that lyophilizing but cannot suppress bacterial fungus etc. to cause.

In RNA coupled reaction, in reverse transcription and pcr amplification reaction, the exRNA Sample adding trehalose is found, do not interfere with the active reaction of various polymerase, ligase and reverse transcription, simultaneously because the trehalose of high concentration is it can be avoided that the formation of RNA higher structure, reaction efficiency can be improved than conventional buffers in reverse transcription and coupled reaction.In addition, the exRNA sample after lyophilizing dissolving, when determined by ultraviolet spectrophotometry, does not affect the reading of OD230/260 and OD260/280；When fluorescence spectrometry exRNA concentration of specimens and integrity, trehalose does not affect fluorescent dye and the combination of exRNA strand, such that it is able to carry out the collection of corresponding data with Qubit 3.0 exometer and Agilent Bioanalyzer 2100.

ExRNA sample after purification is dissolved in above-mentioned protective agent, and vacuum freeze-drying becomes solid-state, at room temperature preserve at least 1 month.

ExRNA sample after purification is dissolved in above-mentioned protective agent, and vacuum freeze-drying becomes solid-state, under the conditions of 2 DEG C-8 DEG C, preserve at least 6 months.

ExRNA sample after purification is dissolved in described protective agent, and vacuum freeze-drying becomes solid-state, preserve under the conditions of-20 DEG C at least 18 months.

In sum; owing to have employed technique scheme; the thermos have the advantages that the protective agent using the present invention; exRNA sample is stored under normal temperature condition can completely preserve 1 month with solid-state; can completely preserve 6 months under the conditions of 2 DEG C-8 DEG C, can completely preserve at least 18 months under the conditions of low temperature-20 DEG C.Under such protective condition, even if multigelation extracellular rna sample still has the highest biological activity, it is to avoid the damage of the exRNA sample that liquid storage exRNA sample multigelation causes.Simultaneously in the presence of this protective agent; do not interfere with the follow-up various activity of enzyme reaction relevant with exRNA; and owing to the chemical property of trehalose can improve exRNA structural stability and improve activity of enzyme reaction; in addition, the trehalose of 0.27M-0.5M do not affect such as ultraviolet spectrophotometer, Qubit 3.0 and Agilent bioanalyzer 2100 etc. can be to the digital independent of the bio-instruments of exRNA sample quantitative and qualitative analysis.Can preserve and not affect integrity and the activity of exRNA in solid form for a long time due to ribonucleic acid, this invention has important using value to ribonucleic acid particularly exRNA, the preservation of Microrna the most therein, Reusability and long-distance transport.

Accompanying drawing explanation

Fig. 1 ribonucleic acid concentration comparison diagram that is exRNA under the conditions of commercialization reagent preserves and aqueous trehalose lyophilizing is saved in-20 DEG C after 18 months；

Fig. 2 is exRNA to be preserved and aqueous trehalose lyophilizing is saved under the conditions of-20 DEG C the OD260/280 numerical value comparison diagram that the ultraviolet spectrometry after 18 months measures at commercialization reagent；

Fig. 3 is exRNA to be preserved and aqueous trehalose lyophilizing is saved under the conditions of 2 DEG C-8 DEG C 6 months and polyacrylamide gel electrophoresis results contrast figure after 1 month under room temperature at commercialization reagent；

Fig. 4 is exRNA to be preserved and the concentration comparison diagram of RNA after multigelation during aqueous trehalose lyophilizing Sample preservation at commercialization reagent；

Fig. 5 is the comparison diagram that different activities RNase inhibitor strengthens aqueous trehalose opposing RNase A degradation capability；

Fig. 6 is the comparison diagram that between 0-1M, variable concentrations aqueous trehalose dissolves exRNA ability；

Fig. 7 is the ultraviolet spectrometry measurement result that between 0-1M, aqueous trehalose dissolves RNA；

Fig. 8 is the comparison diagram of polyacrylamide gel electrophoresis display variable concentrations aqueous trehalose protection exRNA effect；

Fig. 9 is the comparison diagram after Qubit RNA fluorescent quantitation shows RNA stable and aqueous trehalose lyophilizing Sample preservation affected exRNA concentration；

Figure 10 is that the 0.5M aqueous trehalose of different volumes and RNA stable affect comparison diagram to reverse transcription reaction efficiency；

Figure 11 is that the Tris adding variable concentrations in 0.27M aqueous trehalose affects comparison diagram to RNA reverse transcription reaction efficiency；

Figure 12 is that the EDTA adding variable concentrations in 0.5M aqueous trehalose affects comparison diagram to RNA reverse transcription reaction efficiency；

Figure 13 is RNA stable and room temperature preservation 1 month after 0.5M aqueous trehalose lyophilizing exRNA, 2 DEG C-8 DEG C exRNA preserved after preserving 18 months for 6 months and-20 DEG C comparison diagrams of PCR reaction success rate in library construction process；

Figure 14 is in exRNA degradation experiment, the comparison diagram of miR-21 and let-7a molecule copy number change；

Figure 15 is the electrophoretic analysis result figure of exRNA sample after trehalose lyophilizing sample transport；

Figure 16 be Agilent Bioanalyzer 2100 display trehalose lyophilizing sample transport after the content analysis figure of Microrna in sample.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage become apparent from, below in conjunction with embodiment, the present invention is further elaborated.Specific embodiment described herein is merely to illustrate the present invention, is not used in the range limiting the present invention.

The protectant configuration of the various ribonucleic acid of embodiment 1

1. the configuration of variable concentrations aqueous trehalose

Sequence number	Trehalose (g)	Pure water (ml)	Concentration (M)
				1	4.62	50	0.27
2	5.13	50	0.3
				3	6.85	50	0.4
4	8.56	50	0.5
				5	17.11	50	1

Table 1

2. add the aqueous trehalose of ribonuclease inhibitor

Sequence number	Ribonuclease inhibitor (μ l)	Aqueous trehalose (0.5M, μ l)	Volumetric concentration
				1	0.1	2000	1:20000
2	0.2	2000	1:10000
				3	0.4	2000	1:5000
4	1	2000	1:2000

Table 2

3. add ribonuclease inhibitor and the aqueous trehalose of ethylenediaminetetraacetic acid

Table 3

4. add ribonuclease inhibitor and the aqueous trehalose of TE buffer

Table 4

Note: * a.Tris-EDTA buffer is called for short TE buffer (comprising 10mM Tris, 1mM EDTA pH 8.0).

* b. is as regulated the pH value of whole solution so that it is maintained between pH 5.2-8.0, can dissolve mixing realized later by dripping a small amount of concentrated hydrochloric acid or sodium hydroxide.

In the present invention, reagent is if no special instructions, is on market the general reagent that can buy, and described pure water is the purified water without ribonuclease, it is possible to use distilled water.

The purification of embodiment 2 culture of tumor cell exRNA, quantitative determination and integrated degree analysis method:

H332, SW1910 or Hela cell is incubated at the 90mm culture dish of the RPMI culture fluid containing 10%FBS, in 5%CO₂37 DEG C of constant incubators in cultivate the saturation to 90%.Collect cell culture fluid after 48 hours, the culture fluid of every 1ml adds 500 μ l Trizol reagent (LifeTech), repeatedly with left at room temperature after pipettor pressure-vaccum 5 minutes.It is subsequently added the chloroform of 200 μ l, left at room temperature 10 minutes after acutely shaking 15 seconds.12000g draws supernatant after centrifugal 15 minutes in new EP pipe at 4 DEG C, adds isopyknic isopropanol, preserves overnight at-20 DEG C after mixing with pipettor pressure-vaccum.12000g removes supernatant after being centrifuged 15 minutes at 4 DEG C.The 75% ethanol solution concussion adding 500 μ l is washed 1 minute, and 10000g sucks supernatant after being centrifuged 5 minutes at 4 DEG C, is dried the exRNA obtaining purification for 30 minutes under room temperature.According to different experiment purposes exRNA is dissolved in different solutions or lyophilizing is stored under different condition.The solution of the dissolving exRNA chosen has the pure water not containing RNase; the RNA later solution of LifeTech company; the RNA stable solution (preserving exRNA after lyophilizing) of Biomatrica; the aqueous trehalose of variable concentrations, and add the RNase inhibitor of variable concentrations, add the aqueous trehalose (playing protection exRNA effect after lyophilizing) of Tris or EDTA.The quantitative determination ultraviolet spectrophotometer of the exRNA of 10ng/ μ l concentrations above, reading includes OD230/260 and OD260/280.Concrete assay method is: the exRNA that 1-2 μ l dissolves the solution of exRNA and the dissolving of 1-2 μ l respectively is placed in ultraviolet spectrophotometer, wherein dissolve the solution of exRNA as blank to measure concentration and the purity of exRNA, wherein OD260/280 numerical value represents the purity of exRNA, and the OD260/280 numerical value of Utopian exRNA should be between 1.9-2.1.

Quantitative determination Qubit 3.0 fluorophotometer of the exRNA of the following concentration of 10ng/ μ l.Concrete assay method is: by RNA reagent supporting for Qubit instrument with 1:(50-200) dilution proportion, according to description preparation titer and instrument is calibrated.The exRNA solution of 10 μ l is diluted to the working solution of 40-190 μ l, preserves the concentration obtaining RNA after 2 minutes in Qubit3.0 fluorophotometer under room temperature, and initial exRNA concentration is multiplied by gained after 4-10 according to original dilution ratio.

The long-term experiment preserving exRNA under the conditions of-20 DEG C of the different commercialization protective agent of embodiment 3 and aqueous trehalose

The exRNA purified from SW1910 cell by method described in embodiment 1, is dissolved separately in the RNA later solution of LifeTech company, the RNA stable solution of Biomatrica and 0.5M aqueous trehalose, measures the concentration of exRNA through ultraviolet spectrophotometer.Wherein be dissolved in exRNA concentration in RNA later be 13.7ng/ μ l, OD260/280 be 1.91；The exRNA concentration being dissolved in RNA stable be 17.9ng/ μ l, OD260/280 be 1.97；The exRNA concentration being dissolved in 0.5M aqueous trehalose be 21.2ng/ μ l, OD260/280 be 1.99 (Fig. 1 and 2).The exRNA that exRNA and the 0.5M trehalose preserved by RNA stable preserves is lyophilized into solid-state, after the exRNA that RNA later preserves still is positioned over-20 DEG C of cryopreservation 18 months for liquid, again the exRNA of the lyophilised state pure water without RNase being dissolved to lyophilizing front volume, the exRNA that RNA later preserves recovers to room temperature liquid.Again measure concentration and the purity of exRNA in three kinds of solution with ultraviolet spectrometry, be wherein dissolved in exRNA concentration in RNA later be 3.1ng/ μ l, OD260/280 be 1.73；The exRNA concentration being dissolved in RNA stable be 11.9ng/ μ l, OD260/280 be 1.89；The exRNA concentration being dissolved in 0.5M aqueous trehalose be 19.7ng/ μ l, OD260/280 be 1.98 (Fig. 1 and 2).Wherein being dissolved in RNA later exRNA concentration results and measure scope less than ultraviolet spectrophotometer, illustrate that this preservation method exRNA to low temperature state cannot form protection for a long time, majority exRNA have damaged along with freeze thawing and have degraded.

In FIG, black bar diagram represents the concentration after exRNA initial concentration in RNA later solution and-20 DEG C of preservations in 18 months, Dark grey bar diagram represents the concentration after exRNA initial concentration in RNA stable solution and-20 DEG C of preservations in 18 months, and light grey bar diagram represents the concentration after exRNA initial concentration in 0.5M aqueous trehalose and-20 DEG C of preservations in 18 months.Purity after black circles represents exRNA initial purity in RNA later solution and-20 DEG C of preservations in 18 months in fig. 2, Dark grey square represents the purity after exRNA initial purity in RNA stable solution and-20 DEG C of preservations in 18 months, and light grey triangle represents the purity after exRNA initial purity in 0.5M aqueous trehalose and-20 DEG C of preservations in 18 months.

The lyophilizing in different temperatures and different protective agent of embodiment 4exRNA preserves experiment

The exRNA purified from Hela cell and H332 cell by method described in embodiment 1, it is dissolved in RNA stable solution and the 0.5M aqueous trehalose of Biomatrica respectively, every part of solution is divided into two parts, at respectively lyophilizing preserves 1 month at normal temperatures and 2 DEG C-8 DEG C 6 months, again, after being dissolved with isopyknic pure water without RNase by lyophilized powder subsequently, the polyacrylamide gel electrophoresis (also known as PAGE electrophoresis) carrying out exRNA checks the degraded situation of exRNA in during this period of time.The electrophoresis result of Fig. 3 shows, compare with DNA ladder and draw, (1 and 2 exRNA of exRNA and H332 cell representing Hela cell respectively during RNA stable preserves, a, before b and c represents non-lyophilizing respectively, after lyophilizing, room temperature preserves 1 month and 2 DEG C-8 DEG C 6 months after lyophilizing), all there is degraded in various degree in exRNA；And during 0.5M trehalose preserves, there is not any degraded in exRNA, completely the same before the brightness of RNA band and non-lyophilizing.

Fig. 3 is the PAGE electrophoresis result of exRNA.It is the 100bp plus DNA ladder of 0.1 μ g the most successively, the exRNA of the non-lyophilizing of Hela cell, the RNA stable lyophilizing room temperature preservation exRNA of 1 month, the trehalose lyophilizing room temperature preservation exRNA of 1 month, the exRNA of 6 months at RNA stable lyophilizing 2 DEG C-8 DEG C, the exRNA of 6 months at trehalose lyophilizing 2 DEG C-8 DEG C, the exRNA of the non-lyophilizing of H332 cell, the RNA stable lyophilizing room temperature preservation exRNA of 1 month, the trehalose lyophilizing room temperature preservation exRNA of 1 month, the exRNA of 6 months at RNA stable lyophilizing 2 DEG C-8 DEG C, the exRNA of 6 months at trehalose lyophilizing 2 DEG C-8 DEG C.Deposition condition is: lower 30 minutes of 200V voltage.

After embodiment 5exRNA multigelation in different protective agents, damage results compares

The exRNA of equivalent is dissolved separately in RNA later and 0.5M aqueous trehalose, is respectively 5.8ng/ μ l and 6.0ng/ μ l with the average result of the independent repeated measure of Qubit 3.0 three times.Above two parts of samples are placed in-20 DEG C after ten minutes, and RNAlater solution becomes solid-state, and aqueous trehalose remains as liquid.Again sample is put and within 15 minutes, treat that RNA later solution recovers liquid at room temperature, after repeating this operation 5 times, be again respectively 4.3ng/ μ l and 5.9ng/ μ l with the average result of the independent repeated measure of Qubit 3.0 three times.The most again repeating to count by same measuring method after freeze thawing operates 5 times, average result is respectively 2.1ng/ μ l and 5.9ng/ μ l.As shown in Figure 4, transverse axis is number of freezing and thawing to result, and the longitudinal axis is the concentration of exRNA, and black circles represents and is dissolved in RNA later, and light grey square represents and is dissolved in aqueous trehalose.As can be seen from Figure 4; through 5 times and 10 freeze thawing; the exRNA concentration being dissolved in aqueous trehalose has almost no change; the exRNA concentration being dissolved in RNA later reduces 26% and 64% the most respectively; illustrate that the palliating degradation degree of the most exRNA of multigelation number of times is the most serious, this shows that the multigelation that trehalose is applicable to exRNA for the protective capability of exRNA uses.

Adding RNase inhibitor in embodiment 6 aqueous trehalose can protect exRNA to avoid the Degradation of RNase A

The exRNA of equivalent is dissolved in the 0.5M aqueous trehalose of the RNase inhibitor adding variable concentrations activity, is configured to the solution of 5ng/ μ l after Qubit 3.0 measures.Wherein the addition of RNase inhibitor is 1U, 0.5U and 0.1U respectively.The RNase A of 100pg, 10pg and 1pg is added subsequently the most again in solution.After the incubated at room of 1 hour, with the integrity of PAGE electrophoretic examinations exRNA.From figure 5 it can be seen that add the RNase inhibitor energy overprotection exRNA degraded from RNase A, add the degraded that 0.1U just can stand the RNase A of 10pg.Considering from its usage economy, working concentration just can play good anti-exRNA Degradation at the RNase inhibitor of 0.1U.

Embodiment 7 aqueous trehalose concentration affects the dissolubility of RNA

Pure water-soluble RNA is being not readily dissolved in water after lyophilizing, needs auxiliary with heating or stirring, and these operations may affect the stability of RNA thus cause degraded.Trehalose can make pure water-soluble RNA improve its dissolving after lyophilizing due to its distinctive chemical property.Measure the RNA concentration of this sample with ultraviolet spectrophotometer after 5mg RNA is dissolved in the pure water of 0.1ml, its concentration average out to 15 μ g/ μ l under result display room temperature, OD260/280 is about 2.0 (mensuration of concentration and purity the most independently repeats five times), illustrates that this temperature and normal barometric pressure are issued to the saturation of RNA.null0.1M is used respectively subsequently after this RNA sample lyophilizing，0.2M，0.25M，0.27M，0.45M，0.5M，The each 0.1ml of aqueous trehalose of 0.75M and 1M dissolves respectively，Again measure concentration and the purity (mensuration of concentration and purity the most independently repeats five times) of RNA with ultraviolet spectrophotometer in the identical gentle pressure of room temperature，From Fig. 6 and Fig. 7 it appeared that，It is similar with pure water to the solubility property of RNA less than the aqueous trehalose of 0.27M in concentration，At the concentration aqueous trehalose higher than 0.5M, the dissolubility of RNA is tantamount to the aqueous trehalose of 0.27M-0.5M，And purity is held at about 2.0，Thus illustrate that the aqueous trehalose of 0.27-1M can improve the dissolubility of RNA，And do not affect the concentration of RNA，And the aqueous trehalose of 0.27M-0.5M is more preferably to select，The most economical，And the aqueous trehalose solute effect of 0.5M is optimal.In Fig. 6, transverse axis represents the concentration of aqueous trehalose, and the longitudinal axis represents the concentration of the RNA that ultraviolet spectrophotometer measures；In Fig. 7, transverse axis represents the concentration of aqueous trehalose, and the longitudinal axis represents the OD260/280 numerical value that ultraviolet spectrophotometer measures.

Embodiment 8 aqueous trehalose concentration can affect exRNA and preserve

ExRNA is dissolved in the aqueous trehalose of variable concentrations (aqueous trehalose concentration is respectively 0.1M, 0.2M, 0.25M, 0.27M, 0.45M, 0.5M, 0.75M and 1M), is configured to the solution final vacuum lyophilizing of 20ng/ μ l and places at room temperature 1 month.Again after exRNA being dissolved with the pure water without RNase subsequently, with the palliating degradation degree of PAGE electrophoresis detection exRNA.From Fig. 8 it can be found that, the aqueous trehalose less than 0.27M can not protect exRNA very well, and aqueous trehalose concentration is the lowest, and the degraded of exRNA is the most obvious.Aqueous trehalose higher than 0.27M can protect exRNA to avoid degraded; aqueous trehalose more than 0.5M has similar protection exRNA ability with the aqueous trehalose of 0.27M; and the aqueous trehalose more than 1M is difficult to preparation at normal temperatures; therefore; the aqueous trehalose of the preferred 0.27-1M of the present invention; more preferably, the aqueous trehalose of 0.27M 0.5M is used just to have the ability of good preservation exRNA, and more economical.

The aqueous trehalose of embodiment 90.5M does not affect the concentration of Qubit 3.0 fluorescent quantitation detection exRNA

It is stored in the exRNA in RNA stable and 0.5M aqueous trehalose before lyophilizing, is first settled to 5ng/ μ l, independently repeat five times with Qubit3.0, result shows that the exRNA concentration being dissolved in RNA stable, at 4.77 ± 0.38ng/ μ l, is dissolved in the exRNA concentration of 0.5M trehalose at 4.90 ± 0.23ng/ μ l.The most respectively two parts of sample lyophilizing at room temperature preserving are dissolved with equal-volume pure water after 1 month again, the most again with Qubit 3.0 fluorescent quantitation, exRNA concentration is detected.Result display RNA stable has certain impact to Qubit 3.0 fluorescent quantitation detection exRNA, result is 3.98 ± 0.41ng/ μ l through five independent duplicate detection results, and this fluorescent quantitation is almost no impact by 0.5M trehalose, the result independently repeating to read data for five times is 4.85 ± 0.29ng/ μ l, only low compared with beginning mean concentration by 1.1%, completely in range of error, and RNA stable is low by 17% to the detection initial concentration meansigma methods of this fluorescent quantitation, beyond normal range of error, illustrate cause the verity of subsequent experimental data with this protective agent.Comparing RNA stable in Fig. 9 and impact that fluorescent quantitation is detected by 0.5M trehalose, the longitudinal axis represents the exRNA average readings at Qubit 3.0.

Embodiment 100.5M aqueous trehalose and the RNA stable impact on reverse transcription reaction efficiency

ExRNA reverse transcription is become cDNA by quantitatively all relying on library construction of exRNA.With the reaction condition in the Reverse Transcription box of LifeTech company and description, reaction system contains 10 × RT buffer of 1 μ l, the 2.5mM dNTPs of 1 μ l, the M-MLV reverse transcription of 0.5 μ l, the RNase inhibitor of 0.1 μ l, the exRNA of 1 μ l Random primer, 0.1-1 μ g is also settled to 20 μ l reaction systems.Being separately added in overall reaction system and accounting for cumulative volume is 0%, the 0.5M aqueous trehalose of 5%, 10%, 20%, 50% or RNA stable solution.The reaction system being wherein added without 0.5M aqueous trehalose or RNA stable solution is positive controls.Reverse transcription reaction condition is 60 DEG C and heats 5 minutes, hatches on ice 5 minutes, and 42 DEG C are reacted 30 minutes, and 85 DEG C terminate reaction 5 minutes, finally recover to room temperature.The cDNA of reaction gained carries out quantitative fluorescent PCR reaction further and verifies.SYBR Green Master Mix, 12.5 μ l, the 2 μ l cDNA template of LifeTech company, each 0.5 μ l of forward and reverse primer of 10mM let-7a, the final constant volume of PCR reaction system is at 25 μ l.Concrete quantitative fluorescent PCR course of reaction be 95 DEG C 10 minutes, 95 DEG C 15 seconds, 60 DEG C 10 seconds, 72 DEG C 10 seconds, above three steps circulate 40 times, and each reaction is independently repeated 3 times, last 72 DEG C 5 minutes, 95 DEG C 5 minutes and finally recover to room temperature.The fluorescence intensity of each circulation and last product solubility curve record are in ABI 7500fast quantitative fluorescent PCR working software.Final result for 0, with the response value without 0.5M aqueous trehalose and RNA stable solution for 100%, thus generates 0.5M aqueous trehalose or the reverse transcription reaction efficiency of RNA stable solution of different volumes ratio with the template counts of 36 periods.Find from Figure 10, reverse transcription efficiency is started to produce impact by the RNA stable solution of 5%, the RNA stable solution of more than 20% completely inhibit the carrying out of reverse transcription, 0.5M aqueous trehalose from 5% to 50% is all without the carrying out of suppression reverse transcription, and add the 0.5M trehalose higher than 20% and can also to a certain degree improve the efficiency of reverse transcription, when thus explanation exRNA is as template, the trehalose adding 0.5M does not interferes with reverse transcription efficiency.In Figure 10, transverse axis represents trehalose (light grey circle) or RNA stable (black empty circles) accounts for the percentage ratio of overall reaction system, and the longitudinal axis represents the reverse transcription efficiency after adding trehalose (light grey circle) or RNA stable (black empty circles) and the ratio of positive control.

Embodiment 110.27M aqueous trehalose adds the variable concentrations Tris impact on reverse transcription reaction efficiency

PH value has significantly impact to the hydrolysis of RNA, and strong basicity environment can cause the hydrolysis of nucleic acid, and the solution of acid pH then contributes to protecting the stability of RNA.Tris is the conventional solution composition for stable pH value, and adding Tris in aqueous trehalose may impact the exRNA reverse transcription experiment in downstream.For ease of comparing the Tris impact on reverse transcription reaction efficiency, the aqueous trehalose of 0.27M adds the Tris of variable concentrations, ensure that pH value is (the most permissible between PH5.2-8.0 8.0, the present embodiment takes 8.0), according to the inspection reverse transcription reaction efficiency method in embodiment 9, reverse transcription reaction efficiency is weighed, the same Reverse Transcription box using LifeTech and quantitative fluorescent PCR reagent and supporting ABI 7500fast quantitative real time PCR Instrument thereof with the relative quantification of the molecule amount of the let-7a in the cDNA of synthesis.ExRNA is dissolved in the 0.27M aqueous trehalose adding 0mM, 0.1mM, 0.5mM, 1mM, 10mM, 20mM and 100mM Tris respectively, reverse transcription reaction system and condition, and quantitative fluorescent PCR reaction system and condition are completely the same with embodiment 9.Result still with the template counts of 36 periods for 0, the Tris of variable concentrations impact such as Figure 11 on reverse transcription reaction efficiency.As can be seen from Figure 11, the Tris being added up to 100mM does not produce any impact to reverse transcription, and the carrying out of reverse transcription is not the most affected less than the Tris of 0.1mM, thus illustrate that whether adding Tris does not interferes with the biochemical property of exRNA, more of because the trehalose of 0.27M has been able to the structure of stable exRNA.In Figure 11, transverse axis represents the concentration adding Tris, and the longitudinal axis represents the Ct value of let-7a.

Embodiment 120.27M aqueous trehalose adds the variable concentrations EDTA impact on reverse transcription reaction efficiency

EDTA is the chelating agen of bivalent metal ion, and common nuclease needs bivalent metal ion be catalyzed when hydrolyzing RNA, and therefore EDTA can block bivalent metal ion in the solution free, the hydrolysing activity of blocking-up nuclease.Therefore aqueous trehalose adds EDTA and can improve the protective capability to exRNA.But the exRNA reverse transcription in downstream may be tested and be impacted by the addition of EDTA.For ease of comparing the EDTA impact on reverse transcription reaction efficiency, the EDTA of variable concentrations adds the aqueous trehalose of 0.27M, method according to the inspection reverse transcription reaction efficiency in embodiment 9, reverse transcription reaction efficiency is weighed, the same Reverse Transcription box using LifeTech and quantitative fluorescent PCR reagent and supporting ABI 7500fast quantitative real time PCR Instrument thereof with the relative quantification of the molecule amount of the let-7a in the cDNA of synthesis.ExRNA is dissolved in respectively and adds 0mM, 0.01mM, 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, in the 0.27M aqueous trehalose of the EDTA of 5mM and 10mM, reverse transcription reaction system and condition, quantitative fluorescent PCR reaction system and condition are completely the same with embodiment 9 and 10.Result still with the template counts of 36 periods for 0, the EDTA of variable concentrations impact such as Figure 12 on reverse transcription reaction efficiency.As can be seen from Figure 12, reverse transcription is started to produce inhibitory action by the EDTA adding 0.5mM, after EDTA concentration is higher than 1mM, reverse transcription is produced strong inhibition, and the carrying out of reverse transcription is not affected less than the EDTA of 0.2mM, thus explanation adds EDTA can affect the exRNA reaction efficiency as the reverse transcription of template, wherein the addition of 0.2mM EDTA is marginal value, can form downstream higher than this value and significantly inhibit effect.In Figure 12, transverse axis represents the concentration adding EDTA, and the longitudinal axis represents the Ct value of let-7a.

After embodiment 13 lyophilizing exRNA, the holding time affects PCR in library construction process and reacts success rate

At present research shows the most widely, exRNA in secondary order-checking application can the morbidity progress of predicting tumors, therefore exRNA is prepared as can be used for the library that secondary sequenator reads is very important.During this, the success or not of library construction is played a key effect by the success rate of PCR.By exRNA in embodiment 2 and 3 after lyophilised state returns to pure water solution, the most first it is attached testing to exRNA sample with the single stranded RNA ligase of NEB company, PCR amplification is carried out as primer subsequently with linkers, the success rate of PCR accounts for the ratio of all amplified productions as criterion to amplify the product more than 200bp in each EP pipe, and in each EP pipe, coupled reaction and amplified reaction are independent the most in triplicate.This reading from the gel imaging software analysis of Tian Neng company the PAGE electrophoretogram of pcr amplification product is carried out density and fragment products analyzes gained.As shown in Figure 13, transverse axis represents different protective agent respectively under not lyophilisation condition, and lyophilizing room temperature preservation is after 1 month, after lyophilizing 2 DEG C 8 DEG C preserves 6 months, after lyophilizing-20 DEG C preservation 18 months.The longitudinal axis represents the PCR success rate independently repeated in experiment in the most every EP pipe three times.In the case of non-lyophilizing, PCR success rate is all between 90% 95%, illustrate the normal experimental error of this PCR about 5%, and the PCR success rate after lyophilizing room temperature preservation is less than cryopreservation, wherein-20 DEG C preserve after PCR still can reach more than 90% success rate.Comparing with the aqueous trehalose of 0.5M, the PCR success rate of lyophilizing exRNA in RNAstable is low by about 13% 20%.And the PCR efficiency of exRNA after the aqueous trehalose lyophilizing of 0.5M preserves different time remains to maintain more than 88%, illustrate that trehalose in the development that current secondary order-checking is maked rapid progress, can actually play the effect of long-term preservation sample.

The quantitative study of the exRNA palliating degradation degree in the embodiment 14 different holding time

In actual clinical practice, the most order of magnitude at ng or pg of exRNA sample, common quantitative fluorescent PCR can provide the corresponding information of early diagnosis of tumor, personalized treatment and prognosis to the RNA of pg level.But the most clinically the inappropriate method in preservation or frozen-dried protective is dissolved for trace even trace exRNA, accuracy and the feasibility of qualification result can be directly affected.Clinic is most commonly that to be dissolved in pure water exRNA and is distributed into different volumes and is saved in cryogenic refrigerator.This is as proved in previous experiment, and the exRNA that multigelation content is the lowest can cause physical damnification in various degree until degrading.The specific practice of the present embodiment is, the exRNA being dissolved in the 0.5M aqueous trehalose adding RNase inhibitor is divided into three parts, the exRNA being dissolved in the RNA stable adding RNase inhibitor is divided into three parts, above six parts of exRNA samples are respectively placed in lyophilised state 1 month under room temperature, lyophilised state 6 months lyophilised state 18 months lyophilizing 4 times the most repeatedly at lyophilizing 4 times and-20 DEG C the most repeatedly at 2 DEG C-8 DEG C.With Qubit 3.0, exRNA sample is measured the most afterwards.Result shows that degraded in various degree occurs in the exRNA sample standard deviation of lyophilizing in RNA stable, and wherein the exRNA sample of room temperature preservation is less than the limit of Qubit fluorescent quantitation detection, visible obvious degradation occurs.But Qubit fluorescent quantitation can detect the RNA of very short-movie section, therefore the overall picture degraded can not embody in the reading of Qubit completely, this also illustrates that the quality of exRNA can not only be weighed by concentration, and the integrity of exRNA is also the parameter of very important measurement exRNA mass.

The 0.5M aqueous trehalose that contains RNase inhibitor that we use is it can be avoided that lyophilizing brings to exRNA repeatedly damage.It is also random that RNA is degraded for the exRNA different to fragment length by RNase, therefore the Microrna number in exRNA can accurately reflect the palliating degradation degree of exRNA sample, this be also before embodiment in use in this exRNA of let-7a rich in Microrna as the appraisal standards of each item data.Microrna is that a class has 19-22nt, the quantitative requirement of such RNA its must keep complete structure, any change of sequence all can cause detection Microrna copy number change.All being enriched in the H332 extracellular rna of let-7a and miR-21, such as 1pg in the tumor cell line that we use containing having more than 2000 miR-21 and more than 3500 let-7a, the reduction of the molecular number of this kind of Microrna can indicate that the palliating degradation degree of exRNA.When degradation experiment proceeds to the 7th day, we carry out relative quantitative assay to let-7a and the miR-21 molecular number of exRNA in different protective agents.RNA stable exRNA total amount at room temperature is down to about the 50% of initial amount, and the exRNA total amount at 2 DEG C-8 DEG C is down to about the 65% of initial amount, and the exRNA total amount at-20 DEG C is down to about the 80% of initial amount.And all there is not obvious degradation in different temperatures and the exRNA total amount in the holding time in 0.5M trehalose.Thus the molecule amount of the explanation Microrna concentration than individually reference exRNA more can reflect the bulk property of exRNA.Figure 14 is the change cartogram of the molecule copy number of let-7a and miR-21 in exRNA degradation experiment.

The exRNA of embodiment 15 lyophilizing Microrna content analysis experiment after long-distance transport

After the exRNA of SW1910, H332 and Hela cell purifies respectively through Trizol, it is dissolved in 0.5M aqueous trehalose with the concentration of 10ng/ μ l and is lyophilized into solid-state.This lot sample transports the DKFZ (according in customs and aviation relevant laws and regulations, dry powder sample may be used for air transport and customs clearance) of U.S. UCLA the most in dry powder form to, signs for lasting 23 days altogether from domestic FedEx addressee to DKFZ.The machine analysis in-20 DEG C of refrigerators until on the Agilent Bioanalyzer 2100 of the most tested Sample preservation.The content analysis of Microrna is completed by DKFZ's platform laboratory of UCLA.Electrophoretic analysis result is shown in Figure 15.Microrna content (being used for weighing RNA palliating degradation degree) in 6 exRNA samples of this batch of distance transported across country is shown in Figure 16.In this experiment, agents useful for same is the small RNA chip of Agilent company.As can be seen from Figure 14 Microrna accounts for the content of whole exRNA and is above 50%, meeting and exceeding half in current international mainstream research conclusion exRNA is Microrna, six sample standard deviations that result explanation is sent are the most intact, and thus explanation aqueous trehalose is for international sample is academic or clinical communication is very convenient.

The all documents mentioned in the present invention are incorporated as reference the most in this application, are individually recited as with reference to like that just as each document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.

Claims (12)

1. a ribonucleic acid protective agent, it is characterised in that described ribonucleic acid protective agent is for comprising:

Molar concentration is the trehalose of 0.27-1M,

Pure water surplus, and do not comprise ribonuclease.

Ribonucleic acid protective agent the most according to claim 1, it is characterised in that described trehalose molar concentration is 0.27-0.5M。

Ribonucleic acid protective agent the most according to claim 1, it is characterised in that described protective agent also comprises ribonuclease Inhibitor, described ribonuclease inhibitor is 1:(2000-20000 with the volume ratio of aqueous trehalose).

Ribonucleic acid protective agent the most according to claim 2, it is characterised in that also comprise ribonucleic acid in described protective agent Enzyme inhibitor, described ribonuclease inhibitor is 1:(2000-20000 with the volume ratio of aqueous trehalose).

5. according to ribonucleic acid protective agent described in claim 1-4 any claim, it is characterised in that described protective agent also wraps Containing ethylenediaminetetraacetic acid, the content of described ethylenediaminetetraacetic acid is 0.2mM-0.5mM.

6. according to the ribonucleic acid protective agent described in claim 1-4 any claim, it is characterised in that described protective agent In also comprise buffer, after described buffer adds protective agent, make protectant pH value range between 5.2 8.0.

Ribonucleic acid protective agent the most according to claim 6, it is characterised in that described buffer is TE buffer, described The content of ethylenediaminetetraacetic acid is 0.2mM-0.5mM.

Ribonucleic acid protective agent the most according to claim 5, it is characterised in that described trehalose concentration is 0.5M.

9. a ribonucleic acid protective agent test kit, described test kit includes Claims 1-4 or 8 any claim institute The ribonucleic acid protective agent stated.

10. preserving containing any fragment according to the ribonucleic acid protective agent described in Claims 1-4 or 8 any claim Application in the rna sample of length.

The store method of 11. 1 kinds of ribonucleic acid, it is characterised in that described method includes step:

A) rna sample before purification or after purification is dissolved in ribonucleic acid protective agent described in claim 1-4 or 8；

B) solution that step a) obtains is made lyophilized powder；

C) lyophilized powder that step b) obtains is saved in room temperature and temperature below.

The store method of 12. 1 kinds of ribonucleic acid, it is characterised in that described storage temperature is less than-20 DEG C.