CN106868154A - For the primer of K ras gene expression amounts, standard items and preparation method in quantitative determination mescenchymal stem cell - Google Patents
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Abstract
The invention discloses the primer of K ras gene expression amounts, standard items and preparation method in a kind of mescenchymal stem cell for quantitative determination.The method is comprised the following steps：(1) extraction of total serum IgE and cDNA synthesize；(2) PCR amplifications；(3) plasmid standard is prepared.Primer provided by the present invention and plasmid standard have the advantages that the specific good, degree of accuracy is high, sensitivity is high, repeated strong, absolute quantitation can be carried out to K ras gene expressions copy in mescenchymal stem cell in a short time, the time is not only saved, additionally it is possible to the expression of K ras genes in accurate monitoring mescenchymal stem cell.
Description
Technical field
The invention belongs to biological technical field, and in particular to one kind is for K-ras bases in quantitative determination mescenchymal stem cell
Because of the primer of expression quantity, standard items and preparation method.
Background technology
The entitled Kirsten rat meats tumor gene of K-ras genes, is a member in Ras gene families, it has been found that ras genes man
Race is mainly and is made up of K-ras, H-ras and N-ras three types, outer aobvious containing 15 ' non coding exon and 4 codings
Son, the relative molecular mass of coded product is 2.1 ten thousand, therefore referred to as p21 albumen (ras albumen), all it is distributed on cell membrane and thin
In kytoplasm, the differentiation and proliferation of main regulation cell.
Ras genes are tested from two plants of different latent rat viruses by convertibility retrovirus by Ellis etc.
What clone got.Afterwards by research, K-ras, H-ras, N-ras in Ras genes gradually quilt from a variety of tumours
It was found that.And in numerous studies we have found that K-ras genes and the occurrence and development of Several Kinds of Malignancy have it is close contact,
After including K-ras gene activations, the proliferative disorder or benign tumour of cell can be caused to vicious transformation, its albumen is pernicious swollen
Higher than the position normal structure and benign tumour etc., these all cease manner of breathing with the change of K-ras gene expression doses for expression in knurl
Close.
In recent years, whether to tumour illness base Quality Research, the research of tumor-marker gene, or in regenerative medicine
In the research of the application security of the tumorigenesis Journal of Sex Research and stem cell of stem cell, grinding for oncogene expression is directed to
Study carefully, so the monitoring to K-ras gene expression doses just becomes particularly important.
With the fast development of science and technology, the requirement detected for gene expression dose and standard also more and more higher, often
Rule technology is difficult to meet the requirement studied, and Elisa detections need 1-2 days and exist to repeat sex chromosome mosaicism, and normal PCR needs to take
7-8 hour, the fluctuation that its result is produced due to the influence of enzyme system can be converted to product in the PCR being exponentially increased
Very big difference between amount, and Northern blottings are more long due to taking, detection efficiency is low, it is impossible to detect small gene expression
Change.
The content of the invention
For above-mentioned deficiency of the prior art, the present invention provides a kind of for K- in quantitative determination mescenchymal stem cell
The primer of ras gene expression amounts, standard items and preparation method, can effectively solve detection efficiency of the prior art not high, sensitive
Spend the bad problem of relatively low, accuracy.
The primer of K-ras gene expression amounts in a kind of mescenchymal stem cell for quantitative determination, including following sequence：
Sense primer：5'-TTGATTTGTCAGCAGGACCA-3'；
Anti-sense primer：5'-GAGAGTTTCACAGCATGGACTG-3'；
The preparation method of the plasmid standard of K-ras gene expression amounts in a kind of mescenchymal stem cell for quantitative determination,
Comprise the following steps：
(1) mescenchymal stem cell total serum IgE, and reverse transcription synthesis cDNA are extracted in treatment；
(2) with step (1) gained cDNA as template, K-ras gene specific pieces are expanded using the primer described in claim 1
Section；
(3) recovery purifying amplified fragments, clone's culture, obtain plasmid standard.
Further, reverse transcription reaction system is Total RNA 0.1ng~5 μ g, Oilgo (dT) 1 μ l, 5 in step (1)
×Reaction Buffer 4μl、RiboLock RNase inhibitor(20U/μl)1μl、10mMdNTP MIX 2μl、
RevertAid M-Mil V RT (200U/ μ l) 1 μ l, nuclease-free Water supplies 20 μ l.
Further, reverse transcription reaction condition is 42 DEG C, 60min, 70 DEG C, 5min in step (1).
Further, in step (2) amplification system be the μ l of PrimeSTAR Max Premix 25, the μ l of Primer F 3,
The μ l of Primer R 3, cDNA 100~150ng, nuclease-free Water supply 20 μ l.
Further, amplification reaction condition is the stage 1 in step (2)：95 DEG C, 2min:；Stage 2：95 DEG C, 30s；Stage
3：60 DEG C, 15s；Stage 4：72 DEG C, 15s；Stage 5：The stage 2 is returned to, is circulated 29 times；Stage 6：72 DEG C, 5min.
The plasmid standard that above-mentioned preparation method is prepared.
The application of above-mentioned plasmid standard K-ras gene expression amounts in quantitative determination mescenchymal stem cell.
Further, use the method for K-ras gene expression amounts in plasmid standard quantitative determination mescenchymal stem cell for：
(1) plasmid standard concentration is measured, and its copy number is calculated according to copy number computing formula；
(2) it is gradient dilution plasmid standard with 10 times of concentration, using the primer pair various concentrations described in claim 1
Plasmid standard enters performing PCR detection, obtains the Ct values of various concentrations plasmid standard, and set up standard curve；
(3) mescenchymal stem cell total serum IgE, and reverse transcription synthesis cDNA are extracted in treatment, then with cDNA as template, using right
It is required that the primer described in 1 enters performing PCR detection, the Ct values of testing sample are obtained, further according to the standard curve that step (2) is obtained, i.e.,
The expression quantity of K-ras genes in testing sample can be drawn.
Further, PCR reaction systems are 20 μ l in step (2) and step (3), includingFAST
Universal 2X qPCR Master Mix 10μl、Primer F 0.8μl、Primer R 0.8μl、template 1μl、
The μ l of ROX-low 0.4, nuclease-free Water supply 20 μ l.
Further, PCR detections reaction condition is the stage 1 in step (2) and step (3)：95 DEG C, 3min；Stage 2：95
DEG C, 3s；Stage 3：60 DEG C, 30s；Stage 4：2,39 circulations of return stage.
Beneficial effects of the present invention are：
Primer provided by the present invention and standard items, with the specific good, degree of accuracy it is high, sensitivity is high, repeated strong etc.
Advantage, can carry out absolute quantitation, when not only having saved to K-ras gene expressions copy in mescenchymal stem cell in a short time
Between, additionally it is possible to the expression of K-ras genes in accurate monitoring mescenchymal stem cell, is the marker gene of mescenchymal stem cell
The application of mescenchymal stem cell provides help in research, the research of tumor-marker gene and regenerative medicine.
Brief description of the drawings
Fig. 1 is quantitative fluorescent PCR standard curve；
Fig. 2 is fluorescent quantitation solubility curve.
Fig. 3 is amplified fragments electrophoretogram；
Fig. 4 is plasmid standard Sequencing chromatogram；
Fig. 5 is plasmid standard sequencing result and K-ras gene order comparison results；
Fig. 6 is the fluorescent quantitative PCR curve map of the plasmid standard of gradient dilution；
Specific embodiment
Specific embodiment of the invention is described below, this hair is understood in order to those skilled in the art
It is bright, it should be apparent that the invention is not restricted to the scope of specific embodiment, for those skilled in the art,
As long as in appended claim restriction and the spirit and scope of the present invention for determining, these changes are aobvious and easy to various change
See, all are using the innovation and creation of present inventive concept in the row of protection.
Embodiment 1
The primer of K-ras gene expression amounts in a kind of mescenchymal stem cell for quantitative determination, including following sequence：
According to the sequence information table of K-ras genes in ncbi database, primer is designed；
Sense primer：5'-TTGATTTGTCAGCAGGACCA-3'(SEQ ID No:1)；
Anti-sense primer：5'-GAGAGTTTCACAGCATGGACTG-3'(SEQ ID No:2).
The preparation method of the plasmid standard of K-ras gene expression amounts in a kind of mescenchymal stem cell for quantitative determination,
Comprise the following steps：
(1) mescenchymal stem cell total serum IgE, and reverse transcription synthesis cDNA are extracted in treatment
It is 70%~80% by mescenchymal stem cell culture to degree of converging, is added thereto to trypsase, then rinsed with PBS
After 2~3 times, cell RNA is extracted using TRIzol reagents；0.1 μ g~5 μ g total serum IgEs are taken, using RevertAid First
Strand cDNA Synthesis Kit kits (being purchased from Thermo Scientific companies, article No. K1622) are inverted
Record, synthetic sample cDNA；Wherein, as shown in table 1, response procedures are 42 DEG C, 60min, 70 DEG C, 5min to reverse transcription reaction system.
(2) prepared by standard items
The cDNA of reverse transcription synthesis with step (1) enters performing PCR amplification to K-ras genes of interest fragments as template, and right
Amplified production enters row agarose gel electrophoresis, and detection primer specificity, its result is shown in Fig. 3；Wherein, amplification system is shown in Table 2, amplification
Program is the stage 1：95 DEG C, 2min:；Stage 2：95 DEG C, 30s；Stage 3：60 DEG C, 15s；Stage 4：72 DEG C, 15s；Stage 5：Return
The stage 2 is returned to, is circulated 29 times；Stage 6：72 DEG C, 5min.
The above-mentioned amplified fragments of recovery purifying, and be cloned on pGM-T carriers, after placing 30min on ice, it is added into
In the DH5a competent cells of 50 μ l, 30min is placed in ice, taken out, 42 DEG C of water-bath 90s, 2~3min is placed in ice,
Stand, be added thereto to 950 μ l without Amp LB fluid nutrient mediums, the shaken cultivation 1h under conditions of 37 DEG C, 200r/min,
Obtain nutrient solution.
Take the μ l of above-mentioned nutrient solution 100 and coat and contain Amp (100 μ g/mL), X-Gal (20mg/mL) and IPTG
On the LB Solid agar cultures of (100mmol/L), 37 DEG C be inverted culture 14~16h, then with sterilize after transfer needle picking LB
White single bacterium colony in Solid agar culture, is inoculated in LB fluid nutrient mediums of the 3mL containing Amp, 37 DEG C, 200r/min shaking tables
16~20h of culture, obtains strain, and plasmid standard is extracted further according to Tiangeng plasmid extraction kit operating instruction, while confrontation
Grain standard items are identified, and send the Hua Da to carry out gene sequencing, and its result is shown in Fig. 4, and comparison result is shown in Fig. 5；Pass through BioDrop again
Ultramicron nucleic acid-protein concentration analyzer determines its concentration.
The method that K-ras gene quantification detections are carried out using above-mentioned standard product：
(1) it is gradient dilution plasmid standard with 10 times of concentration, obtains the plasmid standard of various concentrations, then pass through
KAPAFAST Universal 2X qPCR Master Mix kits (are purchased from kapa companies, article No. KK4602)
With above-mentioned primer in ABI ViiATMPlasmid standard in 7 to various concentrations enters performing PCR amplification, and PCR amplification curves are shown in Fig. 6；Instead
System is answered to be shown in Table 3, response procedures are the stage 1：95 DEG C, 3min；Stage 2：95 DEG C, 3s；Stage 3：60 DEG C, 30s；Stage 4：Return
2,39 circulations of stage, 3 repetitions are set per gradient concentration, obtain the Ct values of each concentration plasmid standard, then to utilizing software
ViiATM7 pairs of results are analyzed, and obtain standard curve as shown in Figure 1.
(2) K-ras gene expression amounts detection
Gained sample cDNA carries out fluorescent quantitation as template using above-mentioned primer pair K-ras genetic fragments with step (1)
PCR detects that reaction system is shown in Table 3, and course of reaction is the stage 1：95 DEG C, 3min；Stage 2：95 DEG C, 3s；Stage 3：60 DEG C, 30s；
Stage 4：2,39 circulations of return stage；Sample Ct values are obtained, recycles Ct values and step (1) gained standard curve to calculate, i.e.,
Can obtain testing sample copy number, i.e. K-ras gene expression amounts.
The reverse transcription reaction system of table 1
| Reagent | Consumption |
| Total RNA | 0.1ng-5μg |
| Oilgo(dT) | 1μl |
| Water, nuclease-free | Up to 20μl |
| 5×Reaction Buffer | 4μl |
| RiboLock RNase inhibitor(20U/μl) | 1μl |
| 10mMdNTP MIX | 2μl |
| RevertAid M-Mul V RT(200U/μl) | 1μl |
| Total volume | 20μl |
Table 2PCR amplification systems
| Reagent | Consumption |
| PrimeSTAR Max Premix | 25μl |
| Primer F | 3μl |
| Primer R | 3μl |
| cDNA | nμl(100-150ng) |
| Water, nuclease-free | Up to 20μl |
| Total volume | 20μl |
Table 3PCR reaction systems
Experimental example
The repeatability and accuracy validation of absolute quantitation K-ras gene expression amounts.
(1) negative control is set, while identical plasmid is prepared according to the method for above-mentioned steps (2), through BioDrop
Ultramicron nucleic acid-protein concentration analyzer determines its concentration for 70.06ng/ μ l, then again with 10 times of concentration as gradient carry out it is dilute
Release, according to copy number formula：Copy number (cospies/uL)=6.02 × 1023 (cospies/mol) × plasmid concentration (g/
UL)/plasmid molecule quality (g/mol), obtains 5 parts of preferable copy numbers of Plasmid samples, respectively：2.20063×108、
2.20063×107、2.20063×106、2.20063×105。
(2) the K-ras bases according to the standard curve obtained in above-mentioned steps (two) to negative control, 5 parts of Plasmid samples
Because copy number is calculated, it the results are shown in Table 4.
The comparing result of table 4
As can be seen from Table 4, sample detects that the genes of interest copy number for obtaining is copied with preferable under five extension rates
Number results be consistent, without significant difference, it was demonstrated that the present invention can accurate quantitative analysis genes of interest copy number, and repeatability very well.
The standard curve design parameter of table 5
| Slop | -3.385 |
| Y-znter | 39.612 |
| 0.999 | |
| Eff% | 97.445 |
| Error | 0.038 |
Fig. 1 is canonical plotting of the invention, and its design parameter is shown in Table 5, and it can be seen from table 5, standard curve correlation is
99.9%, close to 1, slope shows that standard curve linearity is very good to amplification efficiency -3.3, quantitative also very accurate.And
The independent result for repeating experiment is carried out to sample it is also seen that quantitative result difference is very small, it was demonstrated that the accuracy of this method and
Sensitivity is all fine.
Fig. 2 is the fluorescent quantitation solubility curve figure of this method, shows that primer and method are respectively provided with good specificity, is also had
There is good repeatability.
As shown in figure 3, PCR primer is the single bands of 143bp, showing the primer of design has good specificity.
SEQUENCE LISTING
<110>Co., Ltd of medical test institute of Chengdu Zhong Chuanqing sections
<120>For the primer of K-ras gene expression amounts, standard items and preparation method in quantitative determination mescenchymal stem cell
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
ttgatttgtc agcaggacca 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
gagagtttca cagcatggac tg 22
Claims (11)
1. in a kind of mescenchymal stem cell for quantitative determination K-ras gene expression amounts primer, it is characterised in that including following
Sequence：
Sense primer：5'-TTGATTTGTCAGCAGGACCA-3'；
Anti-sense primer：5'-GAGAGTTTCACAGCATGGACTG-3'.
2. in a kind of mescenchymal stem cell for quantitative determination the plasmid standard of K-ras gene expression amounts preparation method, its
It is characterised by, comprises the following steps：
(1) mescenchymal stem cell total serum IgE, and reverse transcription synthesis cDNA are extracted in treatment；
(2) with step (1) gained cDNA as template, K-ras gene specific fragments are expanded using the primer described in claim 1；
(3) recovery purifying amplified fragments, clone's culture, obtain plasmid standard.
3. in the mescenchymal stem cell for quantitative determination according to claim 2 K-ras gene expression amounts plasmid control
The preparation method of product, it is characterised in that reverse transcription reaction system described in step (1) be Total RNA 0.1ng~5 μ g,
Oilgo(dT)1μl、5×Reaction Buffer 4μl、RiboLock RNase inhibitor(20U/μl)1μl、
The μ l of 10mMdNTP MIX 2, RevertAid M-Mil V RT (200U/ μ l) 1 μ l, nuclease-free Water supply 20 μ
l。
4. in the mescenchymal stem cell for quantitative determination according to claim 2 K-ras gene expression amounts plasmid control
The preparation method of product, it is characterised in that reverse transcription reaction condition described in step (1) is 42 DEG C, 60min, 70 DEG C, 5min.
5. in the mescenchymal stem cell for quantitative determination according to claim 2 K-ras gene expression amounts plasmid control
The preparation method of product, it is characterised in that amplification system described in step (2) be the μ l of PrimeSTAR Max Premix 25,
The μ l of Primer F 3, the μ l of Primer R 3, cDNA 100~150ng, nuclease-free Water supply 20 μ l.
6. in the mescenchymal stem cell for quantitative determination according to claim 2 K-ras gene expression amounts plasmid control
The preparation method of product, it is characterised in that amplification reaction condition described in step (2) is the stage 1：95 DEG C, 2min:；Stage 2：95
DEG C, 30s；Stage 3：60 DEG C, 15s；Stage 4：72 DEG C, 15s；Stage 5：The stage 2 is returned to, is circulated 29 times；Stage 6：72 DEG C,
5min。
7. the plasmid standard that the preparation method described in any one of claim 2~6 is prepared.
8. the application of the K-ras gene expression amounts in quantitative determination mescenchymal stem cell of the plasmid standard described in claim 7.
9. plasmid standard according to claim 8 K-ras gene expression amounts in quantitative determination mescenchymal stem cell
Using, it is characterised in that use the method for K-ras gene expression amounts in plasmid standard quantitative determination mescenchymal stem cell for：
(1) plasmid standard concentration is measured, and its copy number is calculated according to copy number computing formula；
(2) it is gradient dilution plasmid standard with 10 times of concentration, using the plasmid of the primer pair various concentrations described in claim 1
Standard items enter performing PCR detection, obtain the Ct values of various concentrations plasmid standard, and set up standard curve；
(3) mescenchymal stem cell total serum IgE, and reverse transcription synthesis cDNA are extracted in treatment, then with cDNA as template, using claim
Primer described in 1 enters performing PCR detection, the Ct values of testing sample is obtained, further according to the standard curve that step (2) is obtained, you can
Go out the expression quantity of K-ras genes in testing sample.
10. plasmid standard according to claim 9 K-ras gene expression amounts in quantitative determination mescenchymal stem cell
Using, it is characterised in that PCR reaction systems described in step (2) and step (3) are 20 μ l, includingFAST
Universal 2X qPCR Master Mix 10μl、Primer F 0.8μl、Primer R 0.8μl、template 1μl、
The μ l of ROX-low 0.4, nuclease-free Water supply 20 μ l.
11. plasmid standards according to claim 9 K-ras gene expression amounts in quantitative determination mescenchymal stem cell
Using, it is characterised in that the detection reaction conditions of PCR described in step (2) and step (3) are the stage 1：95 DEG C, 3min；Stage 2：
95 DEG C, 3s；Stage 3：60 DEG C, 30s；Stage 4：2,39 circulations of return stage.
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