CN109536585A - Method, matched reagent box and the application of cell one-step method real-time quantitative PCR - Google Patents
Method, matched reagent box and the application of cell one-step method real-time quantitative PCR Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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 present invention provides a kind of method of cell one-step method real-time quantitative PCR, matched reagent and application, there are also kits.The method of cell one-step method real-time quantitative PCR provided by the invention, method and step are as follows: step a, extracting target cell and cracking Buffer is added, after being placed at room temperature for after mixing, be centrifuged off cell fragment and obtain supernatant；Step b, across introne design is carried out to above-mentioned target cell reference gene, and detects specificity；Step c, it takes the supernatant of step a that reaction system liquid is added and carries out step RT-qPCR detection.Reverse transcription and qPCR reagent and program are combined into a step RT-qPCR.On the one hand the present invention wherein develops a kind of lytic cell and cracks supernatant can be directly used for the cracking buffer of RT-qPCR.This cleavage method, which compares tradition and proposes RNA method, has many advantages, such as that time saving and energy saving, operation difficulty is low, security risk is small, at low cost.The method is applicable to kinds of experiments detection, if monoclonal differential expression compares, albumen or the biological action of antibody on cell etc..
Description
Technical field
The invention belongs to molecule and cell biology, in particular to PCR detections.
Background technique
RT-PCR (Real time-polymerase chain reaction, real-time PCR) is a kind of body based on PCR skill
On the basis of art, developed can in the method for real-time detection gene expression dose, be now widely used for pharmic function identification,
The multiple fields such as signal path research and in-vitro diagnosis, are the most important tools for detecting gene transcription level in cell.With life
Object technology diagnosis and pharmaceuticals industry extensive use, for the limited gene expression difference in multiple patient's cells detection,
The a variety of drug candidates of target spot of the same race are to the detection of certain several Gene Expression and the high transcriptionally active of production macromolecular drug
All to relatively large-scale gene transcription level detection, more stringent requirements are proposed for the highly expressed stable cell line in site.
The RT-PCR method of gene transcription level detection in conventional cell is broadly divided into three steps: first, RNA extracting；The
Two, RNA reverse transcription are at cDNA；Third carries out quantitative pcr amplification by fluorescent dye or sonde method.Wherein RNA, which is extracted, is usually
Trizol handles cell, chloroform, it is also necessary to which a variety of organic reagents such as isopropanol and ethyl alcohol, extracting will be time-consuming several every time
Hour, and agents useful for same has toxicity, since RNA is easier to degrade, there are unstability for experiment.In conclusion environment is not
It is friendly, time-consuming, how at high cost required reagent is, more demanding to operator and cumbersome etc., can for multisample experiment
Feasibility is not strong.RNA extracting simultaneously needs cell quantity more, is even more that can not handle in the case of sample preciousness.
In the market there is also singe-cell PCR or sequencing technologies, these technical difficulty are high, at high cost.The sequencing of two generations can divide
Analyse more genes, but the screening of general drug candidate or transcriptionally active site screened, the gene for needing to detect it is single or
It is less, but sample size is huge, even if two generations sequencing cost is declining, is not also suitable for a small amount of changes in gene expression of large sample
Detection.
Summary of the invention
In order to solve the deficiency of above-mentioned existing various methods, on the one hand the present invention wherein develops a kind of lytic cell and splits
Solution supernatant can be directly used for the cracking buffer of RT-qPCR, the method for cell one-step method real-time quantitative PCR, matched reagent and answer
With there are also kits.This cleavage method compare tradition mention RNA method with time saving and energy saving (only needing 10~20min), operation difficulty it is low,
The advantages that security risk is small, at low cost.The method cracking cell conditioned medium cannot be only used for detection normal quantity cell (1000~
2000) destination gene expression, but also it is suitable for a small amount of even few cells (10~100) sample of detection.The method is applicable
It is detected in kinds of experiments, if monoclonal differential expression compares, albumen or the biological action of antibody on cell etc..
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, this method
Steps are as follows:
Step a, it extracts target cell and cracking Buffer is added, after being placed at room temperature for after mixing, be centrifuged off cell fragment and obtain
To supernatant；
Step b, it takes the supernatant of step a that reaction system liquid is added and carries out step RT-qPCR detection.By reverse transcription and qPCR
Reagent and program are combined into a step RT-qPCR.
It in some embodiments further include across introne design being carried out to above-mentioned target cell reference gene, and detect special
The step of property.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, cracking
Buffer includes water, Tris-Hcl, EDTA, Triton X-100, RNase Inhibitor and DTT.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, cracking
Buffer is prepared by water, comprising 5mM Tris-Hcl (pH 8.0), 1~20mM EDTA, 0.1%~2%Triton X-100,
0.2~2U RNase Inhibitor and 0.1M DTT.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, step a
The step of it is as follows: take cell into EP pipe after extracting target cell and cell count, discarded supernatant after centrifugation and a step is added
Method cracks Buffer, and 5~10min is placed at room temperature for after mixing, and centrifugation removal cell fragment obtains supernatant.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, reactants
It is that liquid (can be purchased comprising 1st Strand cDNA Synthesis SuperMix from Wujiang Alongshore Protein Technology Co., Ltd.
Buy), SYBR qPCR SuperMix Plus (can be bought from Wujiang Alongshore Protein Technology Co., Ltd.), corresponding primer and
DEPC water.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, step c
It can also comprise the following steps, take the supernatant 1ul that the reaction system liquid is added in 8 connecting legs, reaction system liquid is 2ul
1st Strand cDNA Synthesis SuperMix, 5ul SYBR qPCR SuperMix Plus, corresponding primer include just
To with each 0.5ul and 11ul DEPC water of reverse primer, final volume 20ul, carry out a step RT-qPCR detection.
According to an aspect of the present invention, the present invention provides a kind of method of cell one-step method real-time quantitative PCR, target is thin
Born of the same parents are CHO-DG44 cell, and corresponding primer is as follows:
CHOGAPDH180-F such as SEQ No.1:GGTTGTCTCCTGCGACTTCA；
CHOGAPDH180-R such as SEQ No.2:GGGTGGGCTTCTTACTCCT；
It is 5 ' to 3 ', not specified following sequence is all 5 ' to 3 '.
When target cell is Jurkat cell, corresponding primer is as follows:
HGAPDH207-F such as SEQ No.3；
HGAPDH207-R such as SEQ No.4；
Also include:
HIL2-F such as SEQ No.5；
HIL2-R such as SEQ No.6.
It is in target cell
The CHO DG44 cell (stable cell line that screening produces the CHO DG44 of IL-6 antibody) of IL-6 monoclonal antibody is produced,
Corresponding primer is as follows:
HIL6A-F such as SEQ No.7；
HIL6A-R such as SEQ No.8.
According to an aspect of the present invention, the present invention provides a kind of cracking for cell one-step method real-time quantitative PCR
Buffer includes water, Tris-Hcl, EDTA, Triton X-100, RNase Inhibitor and DTT.
According to an aspect of the present invention, above-mentioned the present invention provides the application of the method for cell one-step method real-time quantitative PCR
The method of any one cell one-step method real-time quantitative PCR is used for the purpose base of the detection of gene transcription level in cell, cell
Because of the detection of differential expression between the detection of the biological action of the survey inspection of expression, albumen or antibody on cell, monoclonal.
According to an aspect of the present invention, the present invention provides cell one-step method real time quantitative PCR detecting reagent kit, the reagents
Box includes cracking Buffer and reaction system liquid, cracking Buffer include water, Tris-Hcl, EDTA, Triton X-100,
RNase Inhibitor and DTT, reaction system liquid include 1st Strand cDNA Synthesis SuperMix, SYBR
QPCR SuperMix Plus, corresponding primer and DEPC water.
The method of cell one-step method real-time quantitative PCR of the invention is cell Direct Pyrolysis qPCR detection method, abbreviation cell
Direct Pyrolysis method.
The present invention overcomes the cumbersome RNA extraction steps of conventional method, and by RNA extracting, reverse transcription and quantitative PCR
It is combined together, improves efficiency, while result and conventional method have stronger consistency.
Detailed description of the invention
Fig. 1, to be RT-qPCR and cell Direct Pyrolysis q of the invention after extracting RNA in one of the invention embodiment
PCR detection method result-amplification curve and melting curve compare figure；
Fig. 2, for present invention cell Direct Pyrolysis method canonical plotting of the invention in one embodiment；
Fig. 3, for the present invention, cell Direct Pyrolysis method detects blinatumomab antibody pair in one embodiment
Jurkat cell activation；
Fig. 4, ELSIA method detect blinatumomab antibody to Jurkat cell activation；
The RT-qPCR result figure of Fig. 5, Direct Pyrolysis method of the invention；
It Fig. 6, is the present invention in one embodiment after monoclonal cell culture 5 days, 15%SDS-PAGE gel compares
Protein expression difference figure.
Specific embodiment
Following embodiments is some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.The present invention will be further explained below with reference to the accompanying drawings.
The cell Direct Pyrolysis method of the invention of embodiment 1 is compared with tradition RNA method for extracting
1.1 cell Direct Pyrolysis qPCR detection
Using DEPC water prepare 1 × cracking Buffer:5mM Tris-Hcl (pH 8.0)+1~20mM EDTA+0.1%~
2%Triton X-100+0.2~2U RNase Inhibitor (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.)+
0.1M DTT.Recovery CHO-DG44 cell, secondary culture 3 times.It takes cell into EP pipe after cell count, is discarded after centrifugation
Clearly.1 × cracking Buffer is added (final cell densities are 300/ul)；5~10min is placed at room temperature for after mixing, centrifugation removal is thin
Born of the same parents' fragment.Across introne design of primers is carried out to CHO-DG44 cell reference gene gapdh using 5.0 software of Primer, and is made
Its specificity is detected with Blast.It takes 1ul supernatant in 8 connecting legs, 2ul is added1st Strand cDNA
Synthesis SuperMix (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.), 5ul SYBR qPCR
(forward and reverse primer is each for SuperMix Plus (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.), corresponding primer
0.5ul, see Table 1 for details for primer sequence) and 11ul DEPC water, final volume 20ul.Carry out step RT-qPCR detection, 3, each sample
In parallel.Reaction temperature and time such as table 2.
The primer that 1. cell cracking method of table uses compared with tradition RNA extraction process
The detection of 1.2 tradition RNA method for extracting
Use phenol chloroform method extracting 1.0 × 106After a Chinese hamster ovary celI RNA, RNA precipitate is resuspended in the DEPC water of 40ul.Make
With 2% agarose gel identification it is errorless after measure its concentration.0.1ul RNA solution is taken to detect for a step RT-qPCR.(modification is standby
Note: being combined into a step RT-qPCR method for reverse transcription and qPCR, and adjustment number is to match subsequent calculating) reaction time and temperature be same as above.
The specific reaction temperature of table 2.qPCR and time
RT-qPCR and Direct Pyrolysis method qPCR result-amplification curve and melting curve after extraction RNA as shown in Figure 1, wherein
Fig. 1-A is amplification curve, and Fig. 1-B is melting curve.It is that 1. number curve, Direct Pyrolysis method are 2. number curve that tradition, which extracts RNA,.
Fig. 1-B display tradition extracts RNA method and Direct Pyrolysis method melting curve peak value is single, illustrates that the two amplified production is special
It is different and identical.Fig. 1-A amplification curve illustrates traditional RNA extraction process (Ct=17.6 ± 0.51,1250 cells) and directly splits
Difference between solution (Ct=20.3 ± 0.16,300 cells), the experimental results showed that cell quantity differs under conditions of about 8 times
(2500/300 ≈ 8.3), the expression difference (2 of target geneΔCt=2^ (CtDirect Pyrolysis method-CtRNA extraction process) it is about 6.7 times, explanation
It is as a result similar when two methods detect identical quantity cell.It demonstrates cell Direct Pyrolysis qPCR detection method of the invention and passes
Uniting, there are stronger consistency for RNA extraction process.
The foundation of 2 Direct Pyrolysis method cell number detection range of embodiment and standard curve
500 it takes the cell that density is 2000/ul and carries out log10 dilution with 1 × PBS, it is as follows to dilute density: 1000,
250,125,62.5,31.25,15.625/ul.10 × cracking buffer lysis at room temperature 5min, supernatant after high speed centrifugation is added
It is detected for a step RT-qCPR, see Table 2 for details for RT-qPCR program.See Table 1 for details for primer sequence.
Cell Direct Pyrolysis method standard curve as shown in Figure 2, wherein abscissa is log10(cell number), ordinate Ct
Value.
This is the experimental results showed that 15~2000 cell cracking supernatants are used equally for RT-qPCR detection and log10(cell number)
It is in a linear relationship with Ct value.Prove that the destination gene expression of the wider cell of Direct Pyrolysis method detectable amount range is horizontal, simultaneously
The also detectable a small amount of even few cells of the method, compare traditional RNA extraction process with greater advantage.
3 Direct Pyrolysis method of embodiment detects blinatumomab to the activation of Jurkat cell
3.1 plating cells and antibody activation
Jurkat cell counts, with 0.3 × 10 after cell centrifugation resuspension6A/ml density spreads 96 orifice plates；The piping and druming of Raji cell
It is counted after uniformly, with 0.3 × 105A/ml density is spread to be covered in the hole of Jurkat in advance.Various concentration is added in plate overnight
Blinatumomab (be purchased from Amgen) antibody, be incubated overnight.
The activation of 3.2 Direct Pyrolysis methods detection blinatumomab antibody
Suspension is drawn after cell piping and druming uniformly into EP pipe, and 10 × cracking buffer of 1/10 volume is added.Lysis at room temperature
5min takes after high speed centrifugation supernatant to carry out step RT-qPCR detection.Use 5.0 software design il-2 of Primer and reference gene
Gapdh carries out specific detection using Blast.See Table 3 for details for specific primer sequence.
3. antibody of table activates Jurkat cell to test the primer
Cell Direct Pyrolysis method detection blinatumomab antibody as shown in Figure 3 is to Jurkat cell activation, figure
For the expression of IL-2 in Jurkat cell after the detection un-activation of Direct Pyrolysis method and activation.Abscissa is blinatumomab
Using concentration, ordinate is the multiple that sample group compares Control group.
The activation of 3.3ELISA method detection blinatumomab antibody
Detect the secretion of Jurkat cell IL-2.After antibody incubation is stayed overnight, cell conditioned medium is drawn into ELISA Plate, 37 DEG C incubate
1 × PBST is cleaned 3 times after educating 1h, closes 1h using 2%BSA.After cleaning 3 times, Anti IL-2 (being purchased from R&D) is added, 37 DEG C incubate
Educate 1h.After cleaning 3 times, 37 DEG C of incubation 1h of ELIAS secondary antibody are added, after cleaning 3 times, TMB, 37 DEG C of colour developing 20min is added.Use 1M
HCl reads light absorption value under 450nm wavelength after terminating reaction.
ELSIA method detection blinatumomab antibody as shown in Figure 4 is to Jurkat cell activation；The detection of ELSIA method
After un-activation and activation in Jurkat cell supernatant IL-2 secretion content.Abscissa is the blinatumomab concentration used,
Ordinate is OD450 reading.
Using Direct Pyrolysis method detection blinatumomab to the activation of Jurkat cell, as a result, it has been found that 2ng/ml
Big degree activation Jurkat cell and the expression of IL-2 can be improved under the conditions of blinatumomab is existing for the Raji cell.Together
When, ELISA detection confirms the correctness of this result.This is the experiment proves that cell Direct Pyrolysis method can be used for detecting albumen or anti-
Biological action of the body to cell.
4 Direct Pyrolysis method of embodiment detects the difference of monoclonal destination protein expression
4.1 monoclonal bed boards and sampling
It takes the CHO DG44 for producing people IL-6 antibody to stablize strain, counts, eccentric cleaning is twice.Adjust cell density to 3-5/
Cell suspension is laid on 96 orifice plates using the volley of rifle fire, totally two plates by hole.Culture after a week, observes cell state and fluid infusion, after two weeks again
Secondary fluid infusion.After about 20 days, every hole takes cell suspension in 8 connecting legs, and 10 × cracking buffer is added, and concussion mixes.Lysis at room temperature
5min。
4.2RT-qPCR detection
It takes 1ul supernatant into 8 connecting legs after high speed centrifugation, 0.5ul~2ul gDNA Purge is added and (is purchased from Wujiang offshore
Protein Science and Technology Ltd.)+0.1~1ul Rnase Inhibitor, DEPC water mends to 5ul, and two, every hole is parallel.Instantaneously
37 DEG C of reaction 30min are after centrifugation to remove genome.2ul is added1st Strand cDNA Synthesis
SuperMix, 4ul DEPC water, 5ulSYBR qPCR SuperMix Plus and 1ul primer are (positive and anti-
To each 0.5ul of primer, see Table 4 for details for specific primer sequence), step RT-qPCR detection is carried out after brief centrifugation.
The RT-qPCR of Direct Pyrolysis method as shown in Figure 5 is as a result, Direct Pyrolysis method detects the difference of expression between monoclonal
It is different.5-A is amplification curve in Fig. 5, and 5-B is melting curve.1. number curve 1-D6,2. number curve is 2-C2,3. number curve is 2-
C11。
Discovery 1-D6,2-C2 and 2-C11 monoclonal expression, which is detected, through Direct Pyrolysis method is much higher than other groups, wherein
1-D6 expression is high.The results show Direct Pyrolysis method can be used for detecting differential expression between monoclonal, and sensitivity is higher.
4. Direct Pyrolysis method of table and tradition RNA extraction process confirmatory experiment the primer
4.4SDS runs glue and detects monoclonal destination protein differential expression
Above-mentioned monoclonal cell is kept sample after amplifying culture, with equal densities (0.3 × 106A/ml) it is laid on 6 orifice plates, 37
DEG C incubator stationary culture 5 days, cell suspension is taken, 3500rpm is centrifuged 15min and collects cell conditioned medium.Prepare 15%SDS-PAGE
Gel takes 20ul supernatant and 5 × Loading of 5ul Buffer (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.), mixes
Boiling water reacts 5min afterwards, takes on 20ul mixed liquor or 5ul Marker (being purchased from Wujiang Alongshore Protein Technology Co., Ltd.)
Sample, testing goal protein expression.
As shown in fig. 6,15%SDS-PAGE gel compares protein expression difference after monoclonal cell culture 5 days.From result
It was found that the expression of 1-D6 monoclonal is apparently higher than other samples.Protein expression assay result is consistent with Direct Pyrolysis method result, this table
The bright mRNA expression that the more multiple monoclonal target gene of Direct Pyrolysis method can be used, to pick out protein expression most
High monoclonal.
The above embodiments are some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.
SEQUENCE LISTING
<110>Wujiang Alongshore Protein Technology Co., Ltd.
<120>method of cell one-step method real-time quantitative PCR, matched reagent box and application
<130> 2018
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 1
ggttgtctcc tgcgacttca 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 2
gggtgggctt cttactcct 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 3
tctgatttgg tcgtattggg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 4
ggaagatggt gatgggattt 20
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 5
accaggatgc tcacatttaa gtttt 25
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 6
gaggtttgag ttcttcttct agaca 25
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 7
cagactgacc agcaacaagc 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>according to the cell design of detection
<400> 8
caggtagccg tcgtagaaca 20
Claims (10)
1. a kind of method of cell one-step method real-time quantitative PCR, which is characterized in that the method comprises the following steps:
Step a, it extracts target cell and cracking Buffer is added, after being placed at room temperature for after mixing, be centrifuged off cell fragment and obtain
Clear liquid；
Step b, it takes the supernatant that reaction system liquid is added and carries out step RT-qPCR detection.
2. a kind of method of cell one-step method real-time quantitative PCR according to claim 1, which is characterized in that the cracking
Buffer includes water, Tris-Hcl, EDTA, Triton X-100, RNase Inhibitor and DTT.
3. a kind of method of cell one-step method real-time quantitative PCR according to claim 2, which is characterized in that the cracking
Buffer is prepared by water, comprising 5mM Tris-Hcl (pH 8.0), 1~20mM EDTA, 0.1%~2%Triton X-100,
0.2~2U RNase Inhibitor and 0.1M DTT.
4. a kind of method of cell one-step method real-time quantitative PCR according to claim 3, which is characterized in that the step a
It can also comprise the following steps: take cell into EP pipe after the extraction target cell and cell count, discarded supernatant after centrifugation
The one-step method cracking Buffer is added, 5~10min is placed at room temperature for after mixing, centrifugation removal cell fragment obtains supernatant.
5. a kind of method of cell one-step method real-time quantitative PCR according to claim 1, which is characterized in that the reaction
System liquid includes 1st Strand cDNA Synthesis SuperMix, SYBR qPCR SuperMix Plus, corresponding primer
With DEPC water.
6. a kind of method of cell one-step method real-time quantitative PCR according to claim 5, which is characterized in that the step b
It can also comprise the following steps, take the supernatant 1ul that the reaction system liquid, the reaction system liquid are added in 8 connecting legs
For 2ul 1st Strand cDNA Synthesis SuperMix, 5ul SYBR qPCR SuperMix Plus, corresponding primer
Comprising each 0.5ul and 11ul DEPC water of forward and reverse primer, final volume 20ul carries out step RT-qPCR detection.
7. a kind of method of cell one-step method real-time quantitative PCR according to claim 6, which is characterized in that the target
Cell is CHO-DG44 cell, then the corresponding primer is as follows:
CHOGAPDH180-F such as SEQ No.1,
CHOGAPDH180-R such as SEQ No.2；
Or the target cell is Jurkat cell, then the corresponding primer is as follows:
HGAPDH207-F such as SEQ No.3,
HGAPDH207-R such as SEQ No.4,
Also include:
HIL2-F such as SEQ No.5,
HIL2-R such as SEQ No.6；
Or the target cell is the CHO DG44 cell for producing IL-6 monoclonal antibody, then the corresponding primer is as follows:
HIL6A-F such as SEQ No.7,
HIL6A-R such as SEQ No.8.
8. a kind of cracking Buffer for cell one-step method real-time quantitative PCR, which is characterized in that the cracking Buffer includes
Water, Tris-Hcl, EDTA, Triton X-100, RNase Inhibitor and DTT.
9. the application of the method for cell one-step method real-time quantitative PCR, which is characterized in that described in claim 1~7 any one
The method of cell one-step method real-time quantitative PCR is used for the destination gene expression water of the detection of gene transcription level in cell, cell
The detection of differential expression between the detection of the biological action of flat survey inspection, albumen or antibody on cell, monoclonal.
10. a kind of cell one-step method real time quantitative PCR detecting reagent kit, which is characterized in that the kit includes cracking
Buffer and reaction system liquid, the cracking Buffer include water, Tris-Hcl, EDTA, Triton X-100, RNase
Inhibitor and DTT, the reaction system liquid include 1st Strand cDNA Synthesis SuperMix, SYBR qPCR
SuperMix Plus, corresponding primer and DEPC water.
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| CN114107449A (en) * | 2020-08-31 | 2022-03-01 | 中山大学孙逸仙纪念医院 | Single-cell in-situ qPCR method |
| CN116716389A (en) * | 2023-04-20 | 2023-09-08 | 芜湖耄智生物科技有限公司 | Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis |
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| CN114107449B (en) * | 2020-08-31 | 2024-04-09 | 中山大学孙逸仙纪念医院 | Single-cell in-situ qPCR method |
| CN116716389A (en) * | 2023-04-20 | 2023-09-08 | 芜湖耄智生物科技有限公司 | Primer group for detecting TRPC6 mRNA level, kit, detection method and application thereof in Alzheimer disease diagnosis |
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