CN102586416A - Reverse transcription product PCR (polymerase chain reaction) system in reverse transcription PCR analysis of gene expression of IVF (in vitro fertilization) single preimplantation embryos - Google Patents
Reverse transcription product PCR (polymerase chain reaction) system in reverse transcription PCR analysis of gene expression of IVF (in vitro fertilization) single preimplantation embryos Download PDFInfo
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- CN102586416A CN102586416A CN2011103628593A CN201110362859A CN102586416A CN 102586416 A CN102586416 A CN 102586416A CN 2011103628593 A CN2011103628593 A CN 2011103628593A CN 201110362859 A CN201110362859 A CN 201110362859A CN 102586416 A CN102586416 A CN 102586416A
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Abstract
The invention provides a reverse transcription product PCR system in the reverse transcription PCR analysis of the gene expression of IVF single preimplantation embryos. The gene expression analysis method includes the following steps: specimen collection and purification; reverse transcription reaction; reverse transcription of a negative control and a positive control; real-time quantitative PCR assay of a reverse transcription product; optimization of PCR conditions by histocyte RNA (ribose nucleic acid); simultaneous amplification of the negative control and the positive control under the conditions; assay of the expression of a target gene by a relative quantification method; and statistic analysis. The invention also provides a PCR system for the real-time quantitative PCR assay of the reverse transcription product. The improved real-time quantitative reverse transcription PCR technique is highly sensitive and accurate, and can produce considerable data. The method can be utilized to make up for the deficiency of Microarray, and has a wide application value in the analysis of the expression of a single key gene in a single cell and IVF scientific research and clinic. The method provides an important monitoring means for the determination of the new conditions of IVF and in vitro embryo culture medium.
Description
Technical field
The present invention relates to a kind of IVF (In Vitro Fertilization; In-vitro fertilization (IVF)) the gene expression analysis method of single pre-implantation embryos; Be particularly related to the reverse transcription product PCR system in a kind of genetic expression of utilization improvement real-time quantitative reverse transcription PCR (Polymerase Chain Reaction, polymerase chain reaction) the single pre-implantation embryos of technical Analysis IVF.
Background technology
On July 25th, 1978, the happy Blang of " test tube " baby (IVF, in vitro fertilization) the Louis silk of first success is born in Britain in the world.From then on, IVF has brought Gospel for the whole world above the Infertile Couples that accounts for 10%.The Britain scientist Robert Margaret Edwards professor of invention IVF is authorized Nobel's medical physiology prize in 2010.Yet the clinical practice of three more than ten years does not significantly improve the success ratio of IVF.As, 2011, U.S. gestation association statistics 35 years old patient IVF success ratio (fecundation index) about 25-30%, and 1997 also about 30%.And the baby due rate is lower.One of them important reasons is the quality of IVF embryo medium.This is one of most important deciding factor of IVF embryo quality.
At present, selecting the blastula embryo of implantation parent mainly is the morphological characteristic according to the embryo.And do not have a kind of special biological marker and monitoring means to confirm nutrient solution and embryo quality so far as yet.Embryo quality and embryologist's experience is closely related.In fact this artificial selection exists potential dangerous.Even because have the embryo of good morphological characteristic still possibly have transgenation and defective.Through regulating plastosome energy metabolism function, brought into play important effect like Sirtuins albumen in the IVF pre-implantation embryos phase.The defective of Sirt3 gene has been induced the expression of tumor suppressor protein trp53 and has been suppressed embryo's growth.If sirt3 gene and trp53 gene be defective simultaneously, can improve embryo's growth.When this situation also is found in independent trp53 genetic flaw, and the embryo shows as normal morphological characteristic.Utilizing the embryo of real-time quantitative reverse transcription PCR (Real-time quantitative reverse transcription polymerase chainsreaction) method detection mouse fertilized egg vitro culture after 72-96 hour to find that tumor suppressor gene trp53mRNA total amount total among one group of embryo is starkly lower than embryo's same period of growth in the body.RNA microarray (RNA Microarray) shows that also one group of IVF blastula embryo of mouse obviously expressed low trp53mRNA than the blastula embryo of growing in the body.What embryos do not know as yet at present among this group embryo has do not express trp53, if the embryo of this trp53 genetic flaw is implanted to parent, follow-on tumour incidence will increase undoubtedly greatly.Microarray demonstration mouse fertilized egg is external to have 114 and 29 abnormal gene expressions respectively at WhittenShi nutrient solution and 96 hours formed blastula embryos of KSOM+ amino acid nutrient solution cultivation.We show 52-102 abnormal gene expression to the analysis of the mouse blastula embryo that grows in present clinical three kinds of IVF nutrient solutions commonly used, and intensity of anomaly is difference very, and has influenced the growth of the cell function and the vitals of many keys.For example: 1. determine the transcription factor of stem cell potential property, Nanog and UTF1; Stem cell division with keep function; 3. the growth of ventricular muscle cell, the growth of alveolar cell, the growth of neuronic differentiation of pallium and cynapse; 4.B lymphocytic differentiation; 5. cell is to the reaction of dna damage; 6. cell is removed the ability of vldl, or the like.Possibly there is defective in this explanation clinical IVF nutrient solution commonly used at present, that is to say that we do not have optimal IVF nutrient solution as yet so far.Wherein chief reason is not have reliable and effective means to monitor the quality of IVF nutrient solution as yet.
Though microarray (Microarray) analysis provides strong tool to analyze thousands of expression of gene in the single experimentation.Yet it also is not suitable for the RNA amount of sample quite little the time (for Microarray, each sample needs 50ng RNA at least usually, and our experience needs 100 above blastula embryos).This can not make Microarray with 100 blastula embryos of human mononuclear IVF case and analyze, and in other words, Microarray can not detect the means of IVF embryo's genetic expression at present as the mankind.Another kind of important situation is that different genes is very big at ovum and the expression-form difference of each pre-implantation embryos phase; Especially when low by the abundance of the rna transcription of cls gene; And its transcripton life-span is shorter again; And this specific character possibly brought into play crucial effect in adjusting pre-implantation embryos phase function, and the gene of this transient expression also is difficult for monitoring with the Microarray analytical technology.
Summary of the invention
For solving the deficiency of RNA Microarray in gene expression analysis, the invention provides a kind of real-time quantitative reverse transcription PCR of improvement, this is a kind of method that effectively is used for analyzing gene in individual cells and the expression of single implantation pre-embryo.The real-time quantitative reverse transcription PCR technological operation of this improvement requires quite high, but very responsive, highly accurate, can produce considerable data simultaneously.Utilize this method, can remedy the deficiency of Microarray, to analyze single key gene the expression of individual cells and the IVF scientific research with using value is very widely arranged clinically.This technology also provides effective means to remove to detect rna transcription of those potential important transient expressions; How to evaluate and explain the result of the gene expression analysis of accomplishing in total RNA pond, be unlikely to cover the important information of the transcripton of those relatively stable expression.
For reaching the object of the invention, the gene expression method that the real-time quantitative reverse transcription PCR that the present invention sets up is analyzed the single implantation pre-embryo of IVF may further comprise the steps:
Specimen collection and purifying;
Reverse transcription reaction;
Reverse transcription negative control and positive control;
Reverse transcription product carries out real-time quantitative PCR and detects;
The using-system cell RNA is optimized the PCR condition;
Under this condition, amplify amplification negative control and positive control simultaneously;
Adopt the method testing goal expression of gene amount of relative quantification; And
Carry out statistical analysis.
The invention allows for the reaction system that the real-time quantitative PCR that utilizes the real-time quantitative reverse transcription PCR to analyze reverse transcription reaction system and reverse transcription product in the gene expression method of the single implantation pre-embryo of IVF detects; The reaction system that described PCR detects is: template (cDNA) 2 μ l; Taq DNA polymerase 0.75IU, primer (5 ' and 3 ') 1 μ M, dNTPs0.5 μ M; DMSO 0.05 μ l, Mg
++2mM, optical dye SYBR green 0.1 μ l, 10x PCR damping fluid 1 μ l adds MilliQ ultrapure water to final volume 10 μ l.
Owing to when carrying out human IVF, can not utilize more zygote and pre-implantation embryos phase embryo to make gene expression analysis.But can from single IVF case, extract several pre-implantation embryos phase embryo and single embryonic cell is made gene expression analysis to assess this case IVF embryo quality, confirm the biological indicator of IVF pre-implantation embryos through the superfine glass pipette.The contriver of present patent application adopting said method has carried out several important transcripton gene expression analysis to present clinical four kinds of IVF nutrient solutions commonly used to pre-implantation embryos phase embryo, and this method will confirm from now on that for us the new IVF and the condition of external embryo culture nutrient solution provide an important monitoring means again.
Description of drawings
Through the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become obvious.Wherein:
Shown in Figure 1 is immunofluorescence technique and Westernblot analysis BCL2 and FOS expression in the mice embryonic before implantation of a specific embodiment of the present invention;
Shown in Figure 2 is the real-time quantitative reverse transcription PCR analysis Bcl2 and the expression of Fos gene in 30 the total RNA of embryo ponds of a specific embodiment of the present invention;
Shown in Figure 3 is the real-time quantitative reverse transcription PCR technology for detection Bcl2 and the expression of Fos gene in single zygote and two-cell stage embryo of the utilization improvement of a specific embodiment of the present invention;
Shown in Figure 4 is that improvement real-time quantitative reverse transcription PCR of the present invention is used for the step synoptic diagram of analyzing gene in the method for individual cells and the expression of single implantation pre-embryo.
Embodiment
As shown in Figure 4; According to the object of the invention; The present invention utilizes the gene expression method of a kind of real-time quantitative reverse transcription PCR analyzing gene of improvement in individual cells and single implantation pre-embryo; Carry out the quality monitoring of IVF embryo medium with this, the said reverse transcription PCR analyzing gene that utilizes comprises in the step of the genetic expression of individual cells and single implantation pre-embryo:
S1, make a collection of specimens: routine clinical making a collection of specimens; PBS solution (phosphate buffer soln) washing through at least three precoolings; With single zygote or pre-implantation embryos or embryonic cell, total volume is 0.1 μ L through the superfine glass pipette, transfers in the middle of the RNA extract of 0.9 μ L; Said extract is to add 0.1IU RNA enzyme inhibitors in 1 * PCR damping fluid, handles the back through liquid nitrogen and collects whole RNA.
The RNA of S2, collection is further purified with RQ1RNase-Free DNase Kit (Australian Promega company produce).Each embryo or embryonic cell RNA add the damping fluid of 0.1 μ L with 0.1IURQ1 RNase-Free DNase, through 37 ℃ following 10 minutes, add 0.1 μ L stop buffer, 95 ℃ following 1 minute.Through this purified RNA can directly be used for reverse transcription or be stored in subzero 80 ℃ subsequent use, length can be laid in to 6 months.
S3, carry out reverse transcription reaction, its reaction system is following:
Milli Q ultrapure water to the final volume 4 μ l that add special processing
Wherein, the reverse transcription reaction condition is following:
S4, under this condition, carry out reverse transcription negative control (not adding ThermoScript II or RNA template) and positive control (is template with histocyte RNA) simultaneously;
S5, reverse transcription reaction product carry out real-time quantitative PCR, and the PCR reaction system is following:
S6, using-system cell RNA are optimized the PCR condition, and wherein different genes primer optimized conditions is differently (to comprise Mg
++And primer concentration, temperature of fusion, annealing temperature etc.).
PCR condition commonly used among the present invention is following:
S7, under this condition, amplify amplification negative control (not adding ThermoScript II or RNA template) and positive control (comprising the Housekeeper gene) simultaneously like Actb and the expression of goal gene in known histocyte
The threshold difference (Δ Ct) of testing goal gene and Housekeeper gene adopts relative quantification 2 among S8, the threshold difference (Δ Ct) that calculates testing goal gene and Housekeeper gene in the histocyte and the embryo
-Δ Δ ctMethod testing goal expression of gene amount.
S9, statistical analysis.
According to above-mentioned method, a typical instance of the present invention is research mouse proto-oncogene BCL2 and the FOS expression at pre-implantation embryos.BCL2 and FOS are the genes of short cell survival, and mouse ovum and zygote are expressed the BCL2 and the FOS albumen of minute quantity, and BCL2 and FOS albumen (as shown in Figure 1) that each phase of pre-implantation embryos is expressed higher level.Conventional real-time quantitative RT-PCR technical Analysis can detect the genetic expression of BCL2 and FOS at ovum and zygote, but can not detect (as shown in Figure 2) at two cells and later pre-implantation embryos phase thereof.Through single pre-implantation embryos phase embryo is implemented the real-time quantitative RT-PCR analysis, two cell stages of only about about 12% are expressed BCL2 and FOS gene (as shown in Figure 3).Using this technology makes us find this two important transcription factors transient expression rule in the pre-implantation embryos phase.
The present invention is not limited to described embodiment, and those skilled in the art still can do some corrections or change, so rights protection scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention promptly openly in the scope.
Claims (1)
1. a reverse transcription PCR is analyzed the reverse transcription product PCR system in the genetic expression of the single pre-implantation embryos of IVF, and said analyzing gene expression method comprises step:
Specimen collection and purifying;
Reverse transcription reaction;
Reverse transcription negative control and positive control;
Reverse transcription product carries out real-time quantitative PCR and detects;
The using-system cell RNA is optimized the PCR condition;
Under this condition, amplify amplification negative control and positive control simultaneously;
Adopt the method testing goal expression of gene amount of relative quantification; And
Carry out statistical analysis;
Wherein carry out real-time quantitative PCR at reverse transcription product and detect in the step, it is following that reverse transcription product carries out the real-time quantitative PCR reaction system: template (cDNA) 2 μ l, Taq DNApolymerase 0.75IU, primer 1 μ M, dNTPs 0.5 μ M, DMSO 0.05 μ l, Mg
++2mM, optical dye SYBR green 0.1 μ l, 10x PCR damping fluid 1 μ l adds MilliQ ultrapure water to final volume 10 μ l.
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| US20100317916A1 (en) * | 2009-06-12 | 2010-12-16 | Scott Jr Richard T | Method for relative quantitation of chromosomal DNA copy number in single or few cells |
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| US20100317916A1 (en) * | 2009-06-12 | 2010-12-16 | Scott Jr Richard T | Method for relative quantitation of chromosomal DNA copy number in single or few cells |
Non-Patent Citations (2)
| Title |
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| 许静洪、张海莲主编: "《生物化学实验指导》", 31 August 2010 * |
| 邵根宝等: "小鼠植入前胚胎MuERV-L基因表达特征研究", 《南京农业大学学报》 * |
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Inventor after: Jin Xingliang Inventor after: Bai Lijun Inventor after: Chang Jing Inventor before: Yu Qiang |
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Application publication date: 20120718 |