CN110317862A - A method of tiny RNA is detected using mammal embryo culture solution - Google Patents
A method of tiny RNA is detected using mammal embryo culture solution Download PDFInfo
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Abstract
The present invention is a kind of method of type and expression quantity for extracting detection RNA, especially extracts the method for micro RNA in the culture solution of detection cell.Various kinds of cell can secrete micro RNA molecule into culture solution during the cultivation process, this method is by may the end of RNA molecule 3 ' in the culture solution of the especially cell of the sample containing RNA and 5 ' end adjunction heads, then reverse transcription reaction is carried out, it is expanded again, the DNA product expanded can also be analyzed using the method that two generations were sequenced, to judge the type for the RNA that cell is secreted into culture solution and the method for expression quantity.Extraction amplification method of the invention can detect RNA in conjunction with the methods of sequencing, easy to operate, and the RNA information in cell can be learned in the case where not destroying archaeocyte.
Description
Technical field
The present invention relates to biomedical and molecular cytobiology fields, and in particular to micro in a kind of extraction and analysis sample
The method of RNA.
Background technique
MRNA is the product that a chain of DNA is transcribed as template, and the hereditary information that mRNA carries can instruct egg
The synthesis of white matter contains a large amount of hereditary information.Therefore the sequence information of mRNA is very important to gene expression is understood.It removes
The mRNA for instructing albumen to synthesize there are also a large amount of template is transcription non-coding RNA in genome.Including various
MicroRNA (miRNA, siRNA, piwiRNA, piRNA, snoRNA, scaRNA, sdRNA, tsRNA etc.), their length is big
About 22-50nt etc., these RNA will affect a variety of life process, including to embryonic development process and stem cell differentiation
Process has adjustment effect.It all can be by the regulation of various microRNAs more than the gene of half.MicroRNA not there is only
In cell interior, meanwhile, microRNA can be also discharged into culture solution by cell by other methods such as excretion bodies.Due to 1)
MRNA includes the information of a large amount of transcript profiles；2) microRNA has relatively good stability, while expression quantity is relatively stable,
RNA is the biomarker of a potential Non-invasive detection.
For example, the Non-invasive detection of test-tube baby embryo is just important application field.China, which is averaged in every 8 couples of men and wives, just to be had
A pair meets with fertility predicament, and infertile man and wife is in rejuvenation trend, and 25 years old to 30 years old patient is in the majority.In currently,
The infertile patient of state is more than the 12.5% of 40,000,000, Zhan Yuling population.In China, supplementary reproduction is needed to help pregnant reproduction age woman
Female about 3,000,000 or so.And unfortunately, this number is still in Nian Shangsheng.Huge test-tube baby's demand is brought
Be high request to tube-test baby techniques.
The process of test-tube baby is respectively ovulation induction, takes ovum, smart, in vitro fertilization, Embryo Culture, embryo is taken to move in order
Plant, Luteal phase support and pregnancy check.Currently, the success rate of test-tube baby is not high, the success from embryo implantation to fetal birth
Rate is about 30%, and low success rate is greatly the reason is that due to caused by underproof fertilized eggs.Thus, the quality of fertilized eggs
It is most important.The method of the quality of the most fundamental judgement fertilized eggs is morphologic judgment method.Further gene level
Detection be to be realized by invasive method, the detection of ivf zygote usually requires that a cell is taken to go out from embryo
Come, then carry out unicellular sequencing to unicellular, so far, scientists find the expression quantity of some specific microRNAs with
The health status of embryo has certain relevance.But such operation may to be born after baby healthy growth have it is negative
The influence in face.Moreover, from taking single celled process to need accurate operation under microscope again in embryo, it may be said that be a consumption
When effort work.In order to solve this problem, people begin through detection embryo medium to judge the health of corresponding embryo
Whether, it is the Non-invasive detection for embryo.Therefore, by the measurement to RNA in culture solution, it is hopeful to increase the accurate of Non-invasive detection
Rate.But due to the RNA limited amount of embryo's release, detection success rate still has a certain distance with Single cell analysis.
For choosing the biomarker of RNA, most efficient method is to having which RNA carries out qualitative, quantitative in culture solution
Measurement.However the technology of extraction and the analysis of existing RNA there is limitations: 1) needing a large amount of starting microRNA；
2) it since the chain of microRNA is very short, obtains microRNA and amplification has difficulties；3) RNA adjunction head joint efficiency mistake
It is low；4) it is only capable of amplification known array RNA.Therefore, need to find micro RNA technology in a kind of extraction and analysis cell culture fluid.
Summary of the invention
It is an object of the invention in view of the deficiency of the prior art, provide a kind of side for extracting the micro RNA of detection
Method, the method detected in particular with cell culture fluid, can be improved obtain RNA especially microRNA and amplification at
Power improves connector link efficiency, improves the detection efficiency of micro RNA, and method of the invention is easy to operate, safety and reliable
Property is higher.
In the first aspect of the present invention, a kind of method for detecting RNA is provided, described method includes following steps:
(1) sample of RNA may be contained by providing；
(2) lysate and RNA digestive enzyme inhibitor is added；
(3) DNA in DNA digestion enzymic digestion sample is added；
(4) rRNA is added and inhibits primer；
(5) 3 ' adapter-primers and RNA ligase is added, is allowed to be connected to the 3 ' ends of RNA in sample；
(6) reverse transcription primer is added；
(7) 5 ' adapter-primers and RNA ligase is added, is allowed to be connected to the 5 ' ends of RNA in sample；
(8) RNA digestive enzyme inhibitor is added and reverse transcriptase carries out reverse transcription；
(9) RNA in RNA digestion enzymic digestion sample is added；
(10) amplimer is added and archaeal dna polymerase carries out pcr amplification reaction；
(11) step 10 after reaction, amplimer and archaeal dna polymerase is added and carries out secondary PCR amplified reaction.
Preferably, the sample containing RNA is the culture solution of cell, and more preferably fertilized eggs are forming 8 cells
Culture is more than or equal to 24 hours culture solutions before preceding or blastula stage.
Preferably, it is that 5.8s rRNA inhibits primer that the RNA, which inhibits primer, and sequence is SEQ ID NO:1,
Preferably SEQ ID NO:6, i.e. primer 3 ' biotin modification of the end containing arm between TEG, more preferably SEQ ID NO:11.
It is described in any of the above-described preference.Most preferably, described.
In another preferred example, described 3 ' sequence of adapter-primer is SEQ ID NO:3, it is highly preferred that being SEQ ID
NO:7, i.e., the 5 of primer SEQ ID NO:7 ' hold and modify for adenylylation, 3 ' ends are dideoxycytidine modification.
In another preferred example, step (6) reverse transcription primer, sequence are SEQ ID NO:8, i.e., life is contained at 5 ' ends
The modification of object element.
In another preferred example, the 5 ' adapter-primers, sequence are SEQ ID NO:9, preferably SEQ ID NO:
10,5 ' ends are containing amido modified.
In another preferred example, the RNA digestive ferment of the step (9) includes any of RNase A and RNase T1 enzyme
Or combinations thereof.
In another preferred example, the amplimer of the step (10) includes positive amplimer and reversed amplimer,
Preferably positive amplimer sequence is SEQ ID NO:3, and reversed amplimer sequence is SEQ ID NO:4.
In another preferred example, the program of step (10) amplified reaction are as follows:
In another preferred example, the program of the secondary amplified reaction of the step (11) are as follows:
In another preferred example, the step (2) is addition 0.01%-0.2%Triton X-100 as lysate, and
Recombinant RNA digestive ferment inhibit enzyme, 37 DEG C incubation 5-60 minutes.
In another preferred example, the step (3) is that DNA digestive ferment I and Tris-HCl, 37 degree of incubation 15- is added
60min。
In another preferred example, the step (4) is that the 5.8s rRNA that sequence is SEQ ID NO:11 is added to press down
Primer processed, 72 DEG C incubation 10-45 minutes.
In another preferred example, the step (5) be added SEQ ID NO:7 3 ' adapter-primer, PEG 8000,
T4RNA ligase 2, truncated KQ, Tris-HCl, MgCl2, DTT, 30 DEG C incubation 3-8 hours；2-5 unit is added later
Recombinant RNA digestive ferment inhibit enzyme, 4 DEG C incubation 5-16 hours.
In another preferred example, the step (6) is the reverse transcription primer that SEQ ID NO:8 is added, 30 DEG C of incubation 5-45
Minute；And/or Lambda excision enzyme, deadenylation enzyme, 37 DEG C incubation 5-45 minutes.
In another preferred example, the step (7) is that the 5 ' adapter-primers that sequence is SEQ ID NO:10 are added, ATP,
T4RNA ligase, Tris HCl, MgCl2, DTT, 37 DEG C incubation 20-60 minutes.
In another preferred example, the step (8) is that Tris-HCl, KCl, DTT, dNTP, recombinant RNA digestive ferment is added
Inhibit enzyme, 42 DEG C incubation 20-75 minutes；Be added reverse transcriptase, 75 DEG C incubation 5-30 minutes.
In another preferred example, the step (9) is that RNA digestive ferment RNase A and RNase T1, Tris-HCl is added,
37 DEG C incubation 5-60 minutes.
In another preferred example, the step (10) is that MgCl is added2, archaeal dna polymerase, dNTP, the positive amplification of addition
Primer sequence is SEQ ID NO:3, and reversed amplimer sequence is SEQ ID NO:4, and mixed liquor is placed in amplification instrument and is carried out
Amplified reaction, amplification program are as follows:
The step (11) be added step (10) after reaction, the former reaction solution in part is sucked out and is placed in new container
In, MgCl is added2, archaeal dna polymerase, dNTP, the amplimer of addition includes positive amplimer and reversed amplimer, forward direction
Amplimer sequence is SEQ ID NO:3, and reversed amplimer sequence is SEQ ID NO:4；And/or mixed liquor is placed in expansion
Increase and is carried out amplification reaction in instrument, amplification program are as follows:
In another preferred example, the method can also include being purified simultaneously to the claim 1-10 sample obtained
It is sequenced, detects the RNA type and expression quantity in sample.
In the second aspect of the present invention, a kind of kit extracted in detection method for above-mentioned RNA is provided, comprising:
(1) one or more recombinant RNA digestive ferments inhibit enzyme；And/or
(2) DNA digestive ferment I；And/or
(3) 5.8s rRNA inhibits primer；And/or
(4) 3 ' adapter-primers；And/or
(5) 5 ' adapter-primers；And/or
(6) reverse transcription primer；And/or
(7) one or more RNA digestive ferments.
The beneficial effects of the present invention are facilitate convenient simplicity, detected without tissue or cell, extract detection
It is high-efficient, it is able to detect that the microRNA etc. that conventional method does not detect.
Experimental procedure
The acquisition of cell culture fluid: obtaining fertilized eggs by monosperm method, cultivates before forming 8 cells or before blastula stage big
In being equal to 24 hours, a certain amount of culture solution is sucked out, such as 9 μ l culture solutions, the starting material as detection.
Be added in 9 μ l culture solutions 3 μ l lysates (0.01%-0.2%Triton X-100,；2-5 unit recombinant RNA
Digestive ferment inhibits enzyme, Takara or NEB；), 37 DEG C incubation 5-60 minutes.
0.01-1 unit DNAse I, NEB is added after step 2 reaction；10-200pmol Tris-HCl, 37 degree of incubations
15-60min。
2 μ l 5.8s rRNAs are added after step 3 reaction and inhibit primer (2-8pmol), such as SEQ ID NO:1 or
Person SEQ ID NO:6, primer 3 ' end can contain or the biotin modification without arm between TEG, 72 DEG C incubation 10-45 minutes.
10-50pmol 3 ' adapter-primer is added after step 4 reaction, such as SEQ ID NO:2, sequence 5 '
ATCTAATTCTCGNNNNNNATGC 3 ' or SEQ ID NO:7, sequence 5 '
RAppTCTAATTCTCGNNNNNNATGC-ddC 3 ', 5 ' hold and modify for adenylylation, 3 ' ends are dideoxycytidine modification；0.1-
0.2μl PEG 8000；10-100 unit T4RNA ligase 2, truncated KQ, NEB； 50-100pmol Tris-HCl；
10-50pmol MgCl2；1-10pmol DTT；2-5 unit recombinant RNA digestive ferment inhibits enzyme, Takara or NEB.The first step
30 DEG C incubation 3-8 hours.Incubation 5-16 hours of 4 DEG C of second step.
50-500pmol reverse transcription primer is added after step 5 reaction, such as SEQ ID NO:8, sequence 5
' biotin-GCATNNNNNNCGAGAATTAGrA 3 ', biotin modification is contained at 5 ' ends；1-5 unit Lambda excision enzyme,
NEB；5-50 unit deadenylation enzyme, NEB.Incubation 5-45 minutes of 30 DEG C of the first step.Incubation 5-45 minutes of 37 DEG C of second step.
20-100pmol 5 ' adapter-primer is added after step 6 reaction, such as SEQ ID NO:9 or SEQ ID NO:
10；1-5pmol ATP；2-10 unit T4RNA ligase, Thermo Fisher；5-50pmol Tris HCl； 1-10pmol
MgCl2；0.1-2pmol DTT.37 DEG C incubation 20-60 minutes.
100-500pmol Tris-HCl is added after step 7 reaction；200-1500pmol KCl；20-100pmol
DTT；1-10pmol dNTP；2-5 unit recombinant RNA digestive ferment inhibits enzyme；50-500 unit reverse transcriptase, Thermo
Fisher carries out reverse transcription.Incubation 20-75 minutes of 42 DEG C of the first step.Incubation 5-30 minutes of 75 DEG C of second step.
0.01-1 unit RNase A, Thermo Fisher is added after step 8 reaction；0.01-2 unit RNase T1,
Thermo Fisher；10-200pmol Tris-HCl, 37 DEG C incubation 5-60 minutes.
10-200pmol MgCl is added after step 9 reaction2；1-50 unit archaeal dna polymerase；1-20pmol dNTP；
0.01-2pmol amplimer may include positive amplimer and reversed amplimer, such as can be respectively SEQ ID
NO:3 and SEQ ID NO:4.Mixed liquor is placed in amplification instrument and is carried out amplification reaction, amplification program are as follows:
Step 10 after reaction, be sucked out 1-5 μ l original reaction solution be placed in new test tube, add 10-100pmol
MgCl2；0.1-10 unit archaeal dna polymerase；1-20pmol dNTP；0.001-2pmol amplimer.Mixed liquor is placed in amplification
It is carried out amplification reaction in instrument, amplification program are as follows:
Amplified production is conventionally purified, and carries out the sequencing of two generations later to identify small point contained in culture solution
The type and expression quantity of sub- RNA.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1 detection method schematic diagram.
The expression quantity of Fig. 2 .miR-372 is 55rpm.
The expression quantity of Fig. 3 .miR-515 is 66rpm.
The expression quantity of Fig. 4 .miR-603 is 82rpm.
The expression quantity of Fig. 5 .miR-191 is 76rpm.
Specific embodiment
Below in conjunction with drawings and examples of the invention, further details of retouch is made to a specific embodiment of the invention
It states.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as J. Pehanorm Brooker
It writes, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or according to proposed by manufacturer
Condition.
In the embodiment of the present invention unless otherwise instructed, agents useful for same and laboratory apparatus and method are as described below:
Cell culture: the method that regular growth culture can be used.The method of blastocyst culture is by intracytoplasmic sperm injection side
Method obtains fertilized eggs, and progress blastaea training in freshly prepd blastocyst culture liquid is transferred to after being cultivated to the 3rd day blastomere phase
It supports.
Main agents, consumptive material: RNA digestive ferment inhibits enzyme, Takara NEB company；DNAse I, NEB；T4RNA connects
Meet enzyme 2, truncated KQ, NEB company；Lambda excision enzyme, NEB company；Deadenylation enzyme, NEB；T4RNA ligase,
Thermo Fisher company or NEB company；Reverse transcriptase, Thermo Fisher；RNase A, Thermo Fisher are public
Department；RNase T1, Thermo Fisher company；Archaeal dna polymerase, NEB company.
Key instrument: centrifuge, Eppendorf 5424R, PCR thermal cycler, Eppendorf Nexus GX2
Illumina NovaSeq 6000 etc..
Reagent, equipment or the service that specification refers to commercially are bought.
Embodiment 1
The method of the present invention is referring to Fig. 1:
The acquisition of embryo medium: fertilized eggs are obtained by monosperm method, training in 24 hours is cultivated before forming blastula stage
9 μ l, the starting material as detection is sucked out in nutrient solution；
3 μ l lysates are added in 9 μ l culture solutions, and (0.15%Triton X-100,3 unit recombinant RNA digestive ferment inhibits
Enzyme, Takara)
0.5 μ l (0.05 unit) DNAse I, NEB is added after step 2 reaction；0.5 μ l (20pmol) Tris-HCl, 37
Degree is incubated for 20min.
2 μ l 5.8s rRNAs are added after step 3 reaction and inhibit primer (4pmol), such as SEQ ID NO:1 or
SEQ ID NO:6, ' end can contain or the biotin modification without arm between TEG, and 72 DEG C are incubated for 30 minutes for primer 3.
0.2 μ l (20pmol), 3 ' adapter-primer, such as SEQ ID NO:2, sequence 5 ' is added after step 4 reaction
ATCTAATTCTCGNNNNNNATGC 3 ' or SEQ ID NO:7, primer 5 ' end are that adenylylation is modified, and 3 ' ends are double deoxidation
Cytidine modification；0.15μl PEG 8000；0.25 μ l (50 unit) T4RNA ligase 2, truncated KQ, NEB；0.4μl
(80pmol)Tris-HCl；0.6μl(15pmol)MgCl2； 0.3μl(2pmol)DTT；The digestion of 0.1 μ l (4 unit) recombinant RNA
Enzyme inhibits enzyme, Takara.30 DEG C of the first step are incubated for 4 hours.4 DEG C of second step are incubated for 12 hours.
2.2 μ l (220pmol) reverse transcription primers are added after step 5 reaction, such as SEQ ID NO:8, sequence 5
' biotin-GCATNNNNNNCGAGAATTAGrA 3 ', biotin modification is contained at the end of primer 5 '；0.6 μ l (3 unit) Lambda
Excision enzyme, NEB；0.2 μ l (10 unit) deadenylation enzyme, NEB.30 DEG C of the first step are incubated for 15 minutes.37 DEG C of second step are incubated for 30
Minute.
It is added 0.5 μ l (50pmol) 5 ' adapter-primer after step 6 reaction, SEQ ID NO:9 or SEQ ID NO:10,
The end of primer 5 ' is containing amido modified；0.2μl(2pmol)ATP；0.5 μ l (5 unit) T4 RNA ligase, Thermo Fisher；
0.2μl(20pmol)Tris HCl；0.4μl(4pmol)MgCl2； 0.4μl(0.4pmol)DTT.37 DEG C are incubated for 60 minutes.
2 μ l (200pmol) Tris-HCl are added after step 7 reaction；2μl(800pmol)KCl；1μl (50pmol)
DTT；0.9μl(3pmol)dNTP；0.1 μ l (4 unit) recombinant RNA digestive ferment inhibits enzyme；1 μ l (200 unit) reverse transcriptase,
Thermo Fisher carries out reverse transcription.42 DEG C of the first step are incubated for 60 minutes.75 DEG C of second step are incubated for 15 minutes.
0.5 μ l (0.05 unit) RNase A, Thermo Fisher is added after step 8 reaction；(0.05 is single by 0.5 μ l
Position) RNase T1, Thermo Fisher；50pmol Tris-HCl, 37 DEG C are incubated for 30 minutes.
10 μ l (50pmol) MgCl2 are added after step 9 reaction；0.5 μ l (1 unit) archaeal dna polymerase, Thermo
Fisher；16μl(16pmol)dNTP；8 μ l (0.08pmol) forward direction amplimer.Mixed liquor is placed in amplification instrument and is expanded
Increase reaction, amplification program are as follows:
Step 10 after reaction, be sucked out 2 μ l original reaction solutions be placed in new test tube, add 10 μ l (50pmol)
MgCl2；0.25 μ l (0.5 unit) archaeal dna polymerase, Thermo Fisher；10μl(20pmol) dNTP；2.5μl
(0.05pmol) reversed amplimer；0.5 μ l (0.005pmol) forward direction amplimer.Mixed liquor is placed in amplification instrument and is carried out
Amplified reaction, amplification program are as follows:
Amplified production is conventionally purified, and carries out the sequencing of two generations later to identify small point contained in culture solution
The type and expression quantity of sub- RNA.
Amplified production is purified using Zymo Research library purification kit or the library NEB purification kit,
Machine carries out the sequencing of two generations using PE150 strategy and is contained in culture solution with identifying on the Illumina Novaseq sequenator later
The type and expression quantity of microRNA.
Conventional RNA extracts detection method: providing cell culture fluid sample, lysate and RNA digestive enzyme inhibitor is added, adds
Enter rRNA and inhibits primer；3 ' adapter-primers and RNA ligase is added, reverse transcription primer is added, be added 5 ' adapter-primers and
RNA ligase, reverse transcription primer carry out reverse transcription, then carry out PCR.
Experimental result:
The data analysis being sequenced by two generations, the experimental method detect the content of 95 kinds of microRNAs in culture solution
And expression quantity, including various microRNAs such as miRNA, siRNA, piwiRNA, piRNA, snoRNA, scaRNA, sdRNA,
TsRNA etc..The following are the examples of the microRNA detected, such as miR-372, miR-515, miR-603 and miR-191.And
Corresponding RNA can not detected using method of the invention.And routine RNA extraction detection method or unicellular RNA is used to mention
Detection method is taken, the content and expression quantity of 5 kinds of microRNAs in culture solution is only detected, miR-372, miR- is not detected
515, miR-603 and miR-191.
Expression quantity after miR-372 optimization is that can't detect before 55rpm optimizes, referring to fig. 2.Table after miR-515 optimization
It can't detect before being 66rpm optimization up to amount, referring to Fig. 3.Expression quantity after miR-603 optimization, which is that 82rpm optimization is preceding, to be detected not
It arrives, referring to fig. 4.Expression quantity after miR-191 optimization is that can't detect before 76rpm optimizes, referring to Fig. 5.
Sequence used is as shown in the table in the present invention:
Note: " N " refers to any base, and TEG-biotin refers to an arm biotin.NH2 refers to amido modified.RApp refers to adenylylation
Modification.DdC refers to that dideoxycytidine is modified.Biotin refers to biotin modification." r " refers to modified rnase.NH2C6 refers to C6 ammonia
Base modification." H " refers to A or C or T base.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>scientific and technological (Beijing) Co., Ltd is visited on top
<120>a kind of method using mammal embryo culture solution detection tiny RNA
<130> P2018-0613
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 76
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<213>artificial sequence
<220>
<223>5.8s rRNA inhibits primer
<220>
<221> misc_feature
<222> (57)..(76)
<223> n is a, c, g, t or u
<400> 1
atcggcaagc gacgctcaga caggcgtagc cccgggagga acccggggcc gcaagtnnnn 60
nnnnnnnnnn nnnnnn 76
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>3 ' adapter-primers
<220>
<221> misc_feature
<222> (13)..(18)
<223> n is a, c, g, t or u
<400> 2
atctaattct cgnnnnnnat gc 22
<210> 3
<211> 52
<212> DNA
<213>artificial sequence
<220>
<223>positive amplimer
<400> 3
aatgatacgg cgaccaccga ctgacagtcg aagtcagtca gacagtccga cg 52
<210> 4
<211> 63
<212> DNA
<213>artificial sequence
<220>
<223>reversed amplimer
<400> 4
caagcagaag acggcatacg agatattcat cgtgactgga gtgcatctga ctcgagaatt 60
aga 63
<210> 5
<211> 76
<212> DNA
<213>artificial sequence
<220>
<223>5.8s rRNA inhibits primer
<400> 5
atcggcaagc gacgctcaga caggcgtagc cccgggagga acccggggcc gcaagtgcgt 60
tcgaagtgtc gatgat 76
Claims (13)
1. a kind of method for extracting detection RNA, which is characterized in that described method includes following steps:
(1) sample of RNA may be contained by providing；
(2) lysate and RNA digestive enzyme inhibitor is added；
(3) DNA in DNA digestion enzymic digestion sample is added；
(4) rRNA is added and inhibits primer；
(5) 3 ' adapter-primers and RNA ligase is added, is allowed to be connected to the 3 ' ends of RNA in sample；
(6) reverse transcription primer is added；
(7) 5 ' adapter-primers and RNA ligase is added, is allowed to be connected to the 5 ' ends of RNA in sample；
(8) RNA digestive enzyme inhibitor is added and reverse transcriptase carries out reverse transcription；
(9) RNA in RNA digestion enzymic digestion sample is added；
(10) amplimer is added and archaeal dna polymerase carries out pcr amplification reaction；
(11) step 10 after reaction, amplimer and archaeal dna polymerase is added and carries out secondary PCR amplified reaction.
2. extracting detection method as described in claim 1, which is characterized in that
The sample containing RNA is the culture solution of cell, and preferably fertilized eggs are cultivated greatly before forming 8 cells or before blastula stage
In the culture solution for being equal to 24 hours.
3. such as the described in any item extraction detection methods of claim 1-2, which is characterized in that
It is that 5.8s rRNA inhibits primer that the RNA, which inhibits primer, and sequence is SEQ ID NO:1, preferably SEQ ID
NO:6, i.e. primer 3 ' biotin modification of the end containing arm between TEG, more preferably SEQ ID NO:11.
4. extraction detection method as described in any one of claims 1-3, which is characterized in that
It is described 3 that ' sequence of adapter-primer is SEQ ID NO:3, preferably SEQ ID NO:7, i.e. primer SEQ ID NO:7's
5 ' hold and modify for adenylylation, 3 ' ends are dideoxycytidine modification.
5. extraction detection method according to any one of claims 1-4, which is characterized in that
Step (6) reverse transcription primer, sequence are SEQ ID NO:8, i.e., biotin modification is contained at 5 ' ends.
6. extraction detection method as described in any one in claim 1-5, which is characterized in that
5 ' the adapter-primers, sequence are SEQ ID NO:9, and preferably SEQ ID NO:10, amino is contained at the end of primer 5 '
Modification.
7. extraction detection method as claimed in any one of claims 1 to 6, which is characterized in that the RNA of the step (9) digests
Enzyme includes any of RNase A and RNase T1 enzyme or combinations thereof.
8. such as the described in any item extraction detection methods of claim 1-7, which is characterized in that the amplimer of the step (10)
Including positive amplimer and reversed amplimer, positive amplimer sequence is SEQ ID NO:3, reversed amplimer sequence
For SEQ ID NO:4.
9. such as the described in any item extraction detection methods of claim 1-8, which is characterized in that step (10) amplified reaction
Program are as follows:
。
10. such as the described in any item extraction detection methods of claim 1-9, which is characterized in that the secondary amplification of the step (11)
The program of reaction are as follows:
。
11. such as extraction detection method of any of claims 1-10, which is characterized in that the step (2) is to be added
0.01%-0.2%Triton X-100 inhibits enzyme as lysate and recombinant RNA digestive ferment, 37 DEG C incubation 5-60 minutes；With/
Or the step (3) is that DNA digestive ferment I and Tris-HCl, 37 degree of incubation 15-60min is added；The step (4) is that sequence is added
Be classified as SEQ ID NO:11 5.8s rRNA inhibit primer, 72 DEG C incubation 10-45 minutes；And/or the step (5) is
Be added SEQ ID NO:7 3 ' adapter-primer, PEG 8000, T4RNA ligase 2, truncated KQ, Tris-HCl,
MgCl2, DTT, 30 DEG C incubation 3-8 hours；Later be added 2-5 unit recombinant RNA digestive ferment inhibit enzyme, 4 DEG C incubation 5-16 hours；
And/or the step (6) be added SEQ ID NO:8 reverse transcription primer, 30 DEG C incubation 5-45 minutes；Lambda excision enzyme,
Deadenylation enzyme, 37 DEG C incubation 5-45 minutes；And/or the step (7) is that the 5 ' connectors that sequence is SEQ ID NO:10 are added
Primer, ATP, T4RNA ligase, Tris HCl, MgCl2, DTT, 37 DEG C incubation 20-60 minutes；And/or the step (8) is
Be added Tris-HCl, KCl, DTT, dNTP, recombinant RNA digestive ferment inhibit enzyme, 42 DEG C incubation 20-75 minutes；Reverse transcriptase is added,
75 DEG C incubation 5-30 minutes；And/or the step (9) is that RNA digestive ferment RNase A and RNase T1, Tris-HCl, 37 is added
DEG C be incubated for 5-60 minutes；And/or the step (10) is that MgCl is added2, archaeal dna polymerase, dNTP, the positive amplimer of addition
Sequence is SEQ ID NO:3, and reversed amplimer sequence is SEQ ID NO:4, and mixed liquor is placed in amplification instrument and is expanded
Reaction, amplification program are as follows:
The step (11) be added step (10) after reaction, the former reaction solution in part is sucked out and is placed in new container, adds
Enter MgCl2, archaeal dna polymerase, dNTP, the amplimer of addition includes positive amplimer and reversed amplimer, forward direction amplification
Primer sequence is SEQ ID NO:3, and reversed amplimer sequence is SEQ ID NO:4；And/or mixed liquor is placed in amplification instrument
It carries out amplification reaction, amplification program are as follows:
。
12. the extraction detection method as described in claim 1-11, which is characterized in that
The method can also include that the claim 1-10 sample obtained is purified and is sequenced, and detect in sample
RNA type and expression quantity.
13. the kit in a kind of extraction detection method for as described in claim 1-12, which is characterized in that
The kit includes:
(1) one or more recombinant RNA digestive ferments inhibit enzyme；And/or
(2) DNA digestive ferment I；And/or
(3) 5.8s rRNA inhibits primer；And/or
(4) 3 ' adapter-primers；And/or
(5) 5 ' adapter-primers；And/or
(6) reverse transcription primer；And/or
(7) one or more RNA digestive ferments.
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