CN106520997A - Single cell gene expressions quantitative analysis method - Google Patents
Single cell gene expressions quantitative analysis method Download PDFInfo
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- CN106520997A CN106520997A CN201611158473.XA CN201611158473A CN106520997A CN 106520997 A CN106520997 A CN 106520997A CN 201611158473 A CN201611158473 A CN 201611158473A CN 106520997 A CN106520997 A CN 106520997A
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- unicellular
- quantitative analysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a single cell gene expressions quantitative analysis method. The method comprises the steps of adding a single cell to a reaction container; pyrolyzing the single cell; adding a reaction system to the reaction container; executing multi-site RT; executing multi-site PCR amplification by taking cDNA obtained by the multi-site RT as a template; and finally by taking a sequence obtained by the multi-site PCR amplification as a substrate, adding a specific primer for executing specific quantitative PCR to analyze an expression quantity of a specific gene in a single cell, wherein the reaction system added to the reaction container comprises a reaction mixture, a primer pool, reverse transcriptase/polymerase and nuclease-free de-ionized water, the primer pool contains at most 500 pairs of primers, and the reaction mixture contains a buffer system, dNTP and a specificity enhancement factor. According to the method, a large amount of gene expression and gene mutation information in the single cell can be effectively obtained, so that high flux and high sensitivity which cannot be realized by general RT-qPCR are realized.
Description
Technical field
The invention belongs to biological technical field, relates in particular to a kind of method of unicellular Quantitative analysis of gene expression.
Background technology
Traditional gene expression analysis method such as qPCR, Microarray, RNA-seq etc., generally with colony's cell to grind
Study carefully object.The result of this alanysis, ignores the heterogeneity in colony's cell.There is no two duplicate cells in the world,
And individual cells are the basic units of life entity genome regulation and control.In recent years, the gene expression analysis technology of individual cell level, into
For worldwide study hotspot.The diagnosis of research and major disease of this technology just to life science, produces revolution
Property impact, change basis and clinical testing basic mode and thinking.
However, existing single cell analysis technology, such as unicellular RNA-seq etc., need complicated techniqueflow, and it is expensive
Instrument and equipment.Common unicellular qPCR reagents on market, are typically only capable to analyze expression of the individual gene in individual cells,
Just do not existed after cell is analyzed.
The content of the invention
For this purpose, we have developed the unicellular Quantitative analysis of gene expression system of a set of simple, practical, sensitive high flux
(SSA systems).Can a step expand up to 500 specific gene sites in unicellular transcript profile.Product after amplification can use
Any quantitative fluorescent PCR system, the gene loci to having expanded carry out qPCR quantitative analyses.
Specifically, present invention employs technical scheme below：
A kind of method of unicellular Quantitative analysis of gene expression, it is characterised in that methods described includes：In reaction vessel
Add unicellular, crack that this is unicellular, reaction system is added in reaction vessel, many site RT is performed, is obtained with many site RT
The cDNA for obtaining is that template performs Multiplex PCR amplification, finally expands the sequence of acquisition as substrate with Multiplex PCR, adds special
Property primer perform specific quantification PCR analyzing the expression of the unicellular middle specific gene, wherein adding in reaction vessel
Reaction system include reactant mixture, primer pond, reverse transcriptase/polymerase and nuclease free deionized water, primer pond Zhong Bao
Containing the at most multipair primer of 500 pairs, wherein the reactant mixture includes buffer system, dNTP and specific enhancer.
Wherein preferably, it is to add in reaction vessel to perform many site RT with the reaction system performed used by Multiplex PCR
The same set of reaction system for entering, and this two reactions are in one step while performing.
Key component reactant mixture in reaction includes following component or comprises the following ingredients：50-100mM
Tris-HCl pH8.5,10-50mM KCl, 10-50mM (NH4)2SO4, 2-5mM MgCl2, appropriate dNTP and specificity strengthen because
Son.Preferably, the buffer system in the reactant mixture includes：50-100mM Tris-HCl pH8.5,10-50mM KCl,
10-50mM(NH4)2SO4, 2-5mM MgCl2, concentration is with end reaction stereometer.
The specific enhancer included in reactant mixture is answering for RT-PCR, pre-PCR detection especially optimization
Mould assembly PCR additives, effectively increase sensitivity and the homogeneity of detection.The specific enhancer is by following component group
Into：0.2-0.4M trehalose+0-0.2M glycine betaine+1%-3% glycerine+0-20mM sorbierite+0-50mM arginine, concentration is with end
Reaction volume meter.In a more preferred case, the specific enhancer in the reactant mixture is comprised the following ingredients：
1%-3% glycerine+0.2-0.4M trehalose+0-25mM arginine, concentration is with end reaction stereometer.
Further, in reaction system, the concentration of each primer is 0.1 μM, the process of reaction be 50 DEG C of 60min, 95 DEG C
3min, then performs 20 circulations, and each circulation is 95 DEG C of 15sec, 60 DEG C of 15min.
In the above-mentioned methods, single celled cracking sealing freezing cracking preferably after reaction system is added, is then centrifuged for
To discharge cellular content.
Further, it is to add unicellular, sealing in reaction vessel after addition reaction system to freeze cracking condition, be placed in-
5 minutes at 80 DEG C, then 3000rpm is centrifuged 2 minutes.
The present invention dexterously make use of SSA reaction systems to realize single celled cracking and up to 500 gene locis
One-step method is expanded in advance.After sufficient amplified production is obtained, just single qPCR quantitative analyses can be carried out to up to 500 genes.
The method effectively can obtain in individual cells lots of genes expression and gene mutation information, realize common RT-qPCR without
High flux and high sensitivity that method is realized.
Had the advantage that using the solution of the present invention：
1st, operating procedure is simple, reproducible；
2nd, sensitivity is high；
3rd, analysis throughput is high, at most up to 500 genes；
4th, analysis cost is low；
5th, compatible all kinds of downstream analysis systems, without expensive Platform Requirements；
6th, it is widely used, it is adaptable to the sample of the sample or 10pg-100ng RNA of 1-10000 cell.
Description of the drawings
Technical schematic diagrams of the Fig. 1 for the solution of the present invention；
Technology Roadmaps of the Fig. 2 for the present invention program；
Fig. 3 to 6 is the amplification curve of qPCR detections before and after optimization；
Fig. 7 is the thermal map result of confirmatory experiment；
Fig. 8 is the thermal map of the testing result of the single cell level of mouse embryo stem cell and l cell.
Specific embodiment
The know-why of the present invention is as illustrated in fig. 1 and 2.Referring to Fig. 1 and Fig. 2, the present invention dexterously make use of SSA reactants
System realizes single celled cracking and the one-step method of up to 500 gene locis is expanded in advance.After sufficient amplified production is obtained, just
Single qPCR quantitative analyses can be carried out to up to 500 genes.The method effectively can obtain a large amount of in individual cells
Gene expression and gene mutation information, realize high flux and high sensitivity that common RT-qPCR cannot realize.
In an exemplary embodiment, the specific experiment step using the solution of the present invention is as follows：
1st, 0.1 μM of analysis primer pond prepares
Mix the primer (to polyhybird 500 pairs, totally 1000 primers) of all prepare analysis 100 μM of 1 μ L afterwards with not containing
The water polishing of nuclease is to 1mL (now final concentration of 0.1 μM of each primer).
2nd, it is pre- to expand
1) RT-PreAmp Master Mix (SSA systems) are prepared
2) sample-adding, pre- amplified reaction
Take in each hole of the 5 μ L connecting legs of RT-PreAmp Master Mix to 8 or 96 orifice plates.Using mouth suction pipe or streaming
Sorter adds unicellular sample in every hole, is placed in -80 DEG C 5 minutes with 8 connecting leg lids or sealed membrane sealing immediately, after taking-up
3000rpm is centrifuged 2min, it is ensured that is immediately placed in PCR instrument after sealing closely and is reacted as follows：
3rd, Sample Dilution
After pre- amplified reaction terminates, each sample adds 20 μ L nuclease frees water (1:5 dilutions), it is vortexed and mixes,
3000rpm is centrifuged 2min.
4th, qPCR analyses (96 hole qPCR instrument)
Mix, 3000rpm centrifugation 2min are reacted immediately as follows：
(amplified production also can be analyzed with 384 orifice plate qPCR instrument, or Fluidigm Biomark)
Come in conjunction with specific embodiments further below to be described in detail the solution of the present invention.Although it will be appreciated that
Specific numerical value or ginseng are given to reaction condition, each relevant parameter in following present specific embodiment, and each embodiment
Number, but person skilled will be appreciated that given numerical value and parameter are merely possible to for the present invention in the industry
The example for illustrating, and the concrete restriction to the present invention is should not be construed as, therefore protection scope of the present invention should not be caused
Limit, protection scope of the present invention is only limited by claims.
Embodiment site more than 1. reverse transcription, pre- amplified conditions optimization (optimization of 2 × reactant mixture) specificity enhancer
Screening and combination
Common RT, pre-PCR reaction system can realize reverse transcription and pre- amplification procedure step by step, but can not realize one
Process of the footwork from cell to dsDNA, more it cannot be guaranteed that the sensitivity of reaction and homogeneity.And the SSA systems in the present invention
The complex steps of traditional handicraft are not only overcome, one-step method reaction is realized and purpose ds DNA is obtained；And in original base
Reaction homogeneity is improve on plinth, it is possible to achieve while expanding the 500 of 1-10000 cell or 10pg-100ng RNA samples
Individual gene.
(1) optimization of detection sensitivity
2 × reactant mixture is included：100-200mM Tris-HCl (pH8.5), 20-100mM KCl, 20-100mM
(NH4)2SO4, 0.4mM dNTP, 4-10mM MgCl2With specific enhancer.According to document report, trehalose can be improved
The heat endurance of enzyme, improves yield, while promoting the amplification of high GC fragments；Glycine betaine can reduce the Tm values of DNA, improve PCR
Amplification efficiency and specificity；Glycerine can increase the heat endurance of enzyme, improve yield, reduce non-specific amplification.In addition, there is science
Family finds that sorbierite and arginine have good effect in terms of protein structure stability is maintained, in order to improve the sensitive of reaction
Degree and homogeneity, the present invention take the lead in testing two kinds of materials of sorbierite and arginine in PCR reacts, and it was found which can be notable
Improve the reactivity of PCR.
Trehalose test concentrations scope is：0-0.8M；The test concentrations scope of glycine betaine is：0-0.4M；Glycerine test
Concentration range is：0-6%；Sorbierite test concentration range be：0-40mM；Arginine test concentration range be：0-
100mM.Present invention additive of 5 kinds of variable concentrations gradients to more than carries out orthogonal, is configured to 324 kinds of 2 × reactant mixtures.
96 pairs of primers related to mouse early embryonic development are used to make 0.1 μM of primer pond：100 μM each primer
Respectively take 1 μ L to mix, totally 192 μ L, 808 μ L nuclease frees water are mended afterwards to 1mL (final concentration of 0.1 μM of each primer).
The mouse hybridoma cell RNA of 0.01pg/ μ l is used as expanding template.RT-PreAmp is prepared by following system
Master Mix, and add 1 μ L RNA samples.
Excellent 8 connecting leg will be added to be placed in PCR instrument reacted as follows：
After pre- amplified reaction terminates, each sample adds 20 μ L nuclease frees water (1:5 dilutions), it is vortexed and mixes,
3000rpm is centrifuged 2min, takes 5ul and adds 45ul DDW further to dilute (1:10 dilutions), it is vortexed and mixes, 3000rpm centrifugations
2min。
QPCR analyzes (384 hole qPCR instrument)
Mix, 3000rpm centrifugation 2min are reacted immediately as follows.
QPCR reactions terminate post analysis and compare the Ct values and solubility curve of 324 kinds of 2 × reactant mixtures, obtain 4 groups it is clever
The all good combination of sensitivity and specificity, respectively：0-6% glycerine+0.4-0.8M trehalose+0-20mM sorbierites, 2%-6% are sweet
Oil+0.4-0.8M trehalose+0-50mM arginine, 0.2-0.8M trehalose+0.2-0.4M glycine betaine+50-100mM arginine,
0.4-0.8M trehalose+20-40mM sorbierite+0-0.2M glycine betaines.
(2) detect the optimization of uniformity
While detection sensitivity is ensured, the present invention also has performance well in detection homogeneity, according to above-mentioned spirit
The result of sensitivity optimization, select sensitivity and the preferable 4 kinds of 2 × reactant mixtures of specificity carry out detecting the comparison of uniformity and
Optimization.
96 pairs of primers related to mouse early embryonic development are used to make 0.1 μM of primer pond：100 μM each primer
Respectively take 1 μ L to mix, totally 192 μ L, 808 μ L nuclease frees water are mended afterwards to 1mL (final concentration of 0.1 μM of each primer).
The MEC RNA of 100pg is used to do 1：10 stepwise dilution, is finally diluted to 0.01pg
RNA.The RNA of per part of dilution takes 1 μ L and mixes 5 μ L RT-PreAmp Master Mix.
Prepare RT-PreAmp Master Mix
Excellent 8 connecting leg will be added to be placed in PCR instrument reacted as follows：
After pre- amplified reaction terminates, each sample adds 20 μ L nuclease frees water (1:5 dilutions), it is vortexed and mixes,
3000rpm centrifugation 2min take 5ul and add 45ul DDW further to dilute (1:10 dilutions), it is vortexed and mixes, 3000rpm centrifugations
2min。
QPCR analyzes (96 hole qPCR instrument)
Mix, 3000rpm centrifugation 2min are reacted immediately as follows.
Fig. 3-6 show the arginic amplification curves of 2%-6% glycerine+0.4-0.8M trehalose+0-50mM, with preferable
Uniformity, amplification efficiency is between 0.9-1.1.
The sensitivity of SSA systems is verified in the dilution experiment of embodiment 2.RNA.
96 pairs of primers related to mouse early embryonic development are used to make 0.1 μM of primer pond：100 μM each primer
Respectively take 1 μ L to mix, totally 192 μ L, 808 μ L nuclease frees water are mended afterwards to 1mL (final concentration of 0.1 μM of each primer).
The MEC RNA of 8ng/ μ l is used to do 1：4 stepwise dilution, is finally diluted to
0.03pgRNA.The RNA of per part of dilution takes 1 μ L and mixes 9 μ L RT-PreAmp Master Mix (SSA systems).
Prepare RT-PreAmp Master Mix (SSA systems)
Excellent 8 connecting leg will be added to be placed in PCR instrument reacted as follows：
After pre- amplified reaction terminates, each sample adds 40 μ L nuclease frees water (1:5 dilutions), it is vortexed and mixes,
3000rpm is centrifuged 2min.
QPCR analyzes (96 hole qPCR instrument)
Mix, 3000rpm centrifugation 2min are reacted immediately as follows.
Referring to thermal map as shown in Figure 7, the thermal map shows that (background Ct=35 is deducted Log2 gene expression amounts to experimental result
The detection Ct values of specific gene).
This result shows：The SSA Quantitative analysis of gene expression systems of the present invention have high sensitivity, are able to detect that
Less than the RNA sample of 1pg, and there is preferable dynamic range.
Embodiment 3. detects the monocell expressing water of mouse embryo stem cell and l cell using SSA systems
It is flat.
1st, prepare primer pond
96 pairs of primers related to mouse early embryonic development are used to make 0.1 μM of primer pond：100 μM each primer
Respectively take 1 μ L to mix, totally 192 μ L, 808 μ L nuclease frees water are mended afterwards to 1mL (final concentration of 0.1 μM of each primer).
2nd, it is pre- to expand
1) RT-PreAmp Master Mix (SSA systems) are prepared
2) sample-adding, pre- amplified reaction
Take in each hole of the 5 μ L connecting legs of RT-PreAmp Master Mix to 8.Mouse embryonic stem is added using mouth suction pipe
The unicellular sample of cell or MEC, immediately sealing are placed in -80 DEG C 2 minutes, and 1min is centrifuged after taking-up,
And be immediately placed in PCR instrument and reacted as follows：
3rd, Sample Dilution
After pre- amplified reaction terminates, each sample adds 20 μ L nuclease frees water (1:5 dilutions), it is vortexed and mixes,
3000rpm is centrifuged 2min.
4th, qPCR analyses (96 hole qPCR instrument)
Mix, 3000rpm centrifugation 2min are reacted immediately as follows：
Experimental result thermal map shown in Figure 8, the thermal map show that (background Ct=35 deducts spy to Log2 gene expression amounts
Determine the detection Ct values of gene), and the monocell expressing analysis of spectrum of mouse embryo stem cell and MEC.
Result above shows that the unicellular Quantitative analysis of gene expression systems of SSA of the present invention can not only be from unicellular water
The flat upper embryonic stem cell for distinguishing mouse and the fibroblast of mouse, additionally it is possible to find the heterogeneity in this two classes cell, such as
Heterogeneous expression of the Nanog in embryonic stem cell.
Embodiments of the present invention are described in detail above in conjunction with accompanying drawing and instantiation, but the present invention is not limited
In above-mentioned embodiment, in the ken that art those of ordinary skill possesses, can be with without departing from this
Make a variety of changes on the premise of invention objective.
Claims (8)
1. a kind of method of unicellular Quantitative analysis of gene expression, it is characterised in that methods described includes：Add in reaction vessel
Enter unicellular, crack that this is unicellular, reaction system is added in reaction vessel, many site RT are performed, is obtained with many site RT
CDNA be template perform Multiplex PCR amplification, finally with Multiplex PCR expand obtain sequence as substrate, add specificity
Primer performs specific quantification PCR to analyze the expression of the unicellular middle specific gene, wherein add in reaction vessel
Reaction system includes reactant mixture, primer pond, reverse transcriptase/polymerase and nuclease free deionized water, and primer is included in pond
The at most multipair primer of 500 pairs, wherein the reactant mixture includes buffer system, dNTP and specific enhancer.
2. the method for unicellular Quantitative analysis of gene expression as claimed in claim 1, it is characterised in that perform many site RT with
The reaction system performed used by Multiplex PCR is the same set of reaction system added in reaction vessel, and this two reactions
It is in one step while performing.
3. the method for unicellular Quantitative analysis of gene expression as claimed in claim 1, it is characterised in that the reaction mixing
Buffer system in thing includes：50-100 mM Tris-HCl pH8.5,10-50 mM KCl, 10-50 mM (NH4)2SO4, 2-
5 mM MgCl2, concentration is with end reaction stereometer.
4. the method for unicellular Quantitative analysis of gene expression as claimed in claim 1, it is characterised in that the reactant mixture
In specific enhancer comprise the following ingredients：0.2-0.4M trehalose+0-0.2M glycine betaine+1%-3% glycerine+0-
20mM sorbierite+0-50mM arginine, concentration is with end reaction stereometer.
5. the method for unicellular Quantitative analysis of gene expression as claimed in claim 4, it is characterised in that the reactant mixture
In specific enhancer comprise the following ingredients：1%-3% glycerine+0.2-0.4M trehalose+0-25mM arginine, concentration
With end reaction stereometer.
6. the method for unicellular Quantitative analysis of gene expression as claimed in claim 2, it is characterised in that every in reaction system
The concentration of one primer is 0.1 μM, and the process of reaction is 50 DEG C of 60min, 95 DEG C of 3min, then performs 20 circulations, each
Circulate as 95 DEG C of 15sec, 60 DEG C of 15min.
7. the method for unicellular Quantitative analysis of gene expression as claimed in claim 1, it is characterised in that single celled cracking is
After reaction system is added, sealing freezing cracking, is then centrifuged for discharging cellular content.
8. the method for unicellular Quantitative analysis of gene expression as claimed in claim 6, it is characterised in that freezing cracking condition is
Unicellular, sealing is added after reaction system is added in reaction vessel, 5 minutes at being placed in -80 DEG C, then 3000rpm is centrifuged 2 points
Clock.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107723343A (en) * | 2017-11-28 | 2018-02-23 | 宜昌美光硅谷生命科技股份有限公司 | A kind of method of gene quantification analysis |
| CN109033743A (en) * | 2018-07-25 | 2018-12-18 | 上海交通大学 | A method of reducing technology noise in unicellular transcript profile data |
| CN109112182A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The method of the single oocyte gene quantitative expression of one boar |
| CN109251965A (en) * | 2017-07-12 | 2019-01-22 | 基因凯斯特有限公司 | A kind of DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102348811A (en) * | 2009-01-13 | 2012-02-08 | 弗卢伊蒂格姆公司 | Single-cell nucleic acid analysis |
| CN102533960A (en) * | 2010-12-31 | 2012-07-04 | 深圳华大基因科技有限公司 | Single-cell genome analysis method and kit |
| CN104911274A (en) * | 2015-07-06 | 2015-09-16 | 四川医科大学 | Method for detecting single-celled real-time fluorescence quantitative polymerase chain reaction |
| CN105463125A (en) * | 2016-02-02 | 2016-04-06 | 江苏正大天创生物工程有限公司 | Nucleic acid amplification system and freeze-drying protective agent thereof |
-
2016
- 2016-12-14 CN CN201611158473.XA patent/CN106520997A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102348811A (en) * | 2009-01-13 | 2012-02-08 | 弗卢伊蒂格姆公司 | Single-cell nucleic acid analysis |
| CN102533960A (en) * | 2010-12-31 | 2012-07-04 | 深圳华大基因科技有限公司 | Single-cell genome analysis method and kit |
| CN104911274A (en) * | 2015-07-06 | 2015-09-16 | 四川医科大学 | Method for detecting single-celled real-time fluorescence quantitative polymerase chain reaction |
| CN105463125A (en) * | 2016-02-02 | 2016-04-06 | 江苏正大天创生物工程有限公司 | Nucleic acid amplification system and freeze-drying protective agent thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109251965A (en) * | 2017-07-12 | 2019-01-22 | 基因凯斯特有限公司 | A kind of DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity |
| CN109251965B (en) * | 2017-07-12 | 2022-11-18 | 基因凯斯特有限公司 | PCR buffer composition for enhancing DNA polymerase activity with increased gene mutation specificity |
| CN107723343A (en) * | 2017-11-28 | 2018-02-23 | 宜昌美光硅谷生命科技股份有限公司 | A kind of method of gene quantification analysis |
| CN109033743A (en) * | 2018-07-25 | 2018-12-18 | 上海交通大学 | A method of reducing technology noise in unicellular transcript profile data |
| CN109033743B (en) * | 2018-07-25 | 2021-01-01 | 上海交通大学 | Method for reducing technical noise in single-cell transcriptome data |
| CN109112182A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The method of the single oocyte gene quantitative expression of one boar |
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