CN102864211A - Reverse transcription reaction system for analyzing genetic expression of IVF (in-vitro fertilization) single implanting pre-embryo by reverse transcription PCR (polymerase chain reaction) - Google Patents
Reverse transcription reaction system for analyzing genetic expression of IVF (in-vitro fertilization) single implanting pre-embryo by reverse transcription PCR (polymerase chain reaction) Download PDFInfo
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- CN102864211A CN102864211A CN201110362856XA CN201110362856A CN102864211A CN 102864211 A CN102864211 A CN 102864211A CN 201110362856X A CN201110362856X A CN 201110362856XA CN 201110362856 A CN201110362856 A CN 201110362856A CN 102864211 A CN102864211 A CN 102864211A
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Abstract
The invention provides a reverse transcription reaction system for analyzing the genetic expression of an IVF (in-vitro fertilization) single implanting pre-embryo by reverse transcription PCR (polymerase chain reaction). An analysis gene expression method comprises the following steps of: collecting and purifying a specimen; carrying out reverse transcription reaction; carrying out negative control and positive control of the reverse transcription; carrying out real-time quantitative PCR detection on a reverse transcription product; optimizing a PCR condition by tissue cell RNA (ribose nucleic acid); under the condition, simultaneously amplifying and augmenting the negative control and the positive control; detecting the expression amount of a target gene with a relatively-quantitative method; and carrying out statics analysis. The improved real-time quantitative reverse transcription PCR technology is very sensitive and is highly precise, and meanwhile, considerable data can be generated. With the method, the deficiency of Microarray is made up, and the method has very wide application value for analyzing a single key gene on the expression of a single cell as well as the scientific research and the clinics of IVF. With the method, a very important monitoring means is provided for determining a new condition of IVF and culture fluid for in-vitro embryo culture.
Description
Technical field
The present invention relates to a kind of IVF (In Vitro Fertilization, in-vitro fertilization (IVF)) the gene expression analysis analysis method of Single Embryo, be particularly related to the reverse transcription reaction system in a kind of gene expression method of utilization improvement Real Time RT-PCR (Polymerase Chain Reaction, polymerase chain reaction) technical Analysis IVF Single Embryo.
Background technology
On July 25th, 1978, the happy Blang of " test tube " baby of first success (IVF, in-vitro fertilization (IVF)) Louise 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 British scientist Robert Margaret Edwards professor of invention IVF is awarded 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 (conceptual quotient) about 25-30%, and 1997 also about 30%.And the baby due rate is lower.One of them important reason is the quality of IVF embryo medium.This is one of most important deciding factor of IVF embryo quality.
At present, the blastula embryo of selection implantation parent mainly is the morphological characteristic according to the embryo.And not yet have so far a kind of special biological marker and monitoring means to determine nutrient solution and embryo quality.Embryo quality and embryologist's experience is closely related.In fact this artificial selection exists potential danger.Even because have the embryo of good morphological characteristic still may have transgenation and defective.By regulating the mitochondrial function, brought into play important effect in the IVF pre-implantation embryos phase such as Sirtuins albumen.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 RT-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 tumor growth.RNA microarray (RNA Microarray) shows that also one group of IVF blastula embryo of mouse obviously expressed low trp53mRNA than the blastula embryo of tumor growth.What embryos not yet know 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 Tumor incidence will increase undoubtedly greatly.Microarray demonstration mouse fertilized egg is external to have respectively 114 and 29 abnormal gene expressions 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 Mouse Blastocysts phase embryo's that grows in present clinical three kinds of IVF nutrient solutions commonly used analysis, and intensity of anomaly is very different, and has affected the growth of cell function and the vitals of many keys.For example: 1. determine the transcription factor of stem cell potential, Nanog and UTF1; 2. stem cell division and keep function; 3. the growth of ventricular muscle cell, the growth of alveolar cell, the differentiation of Cortical Neurons and the growth of cynapse; 4.B lymphocytic differentiation; 5. cell is to the reaction of dna damage; 6. the ability of cell clearance vldl, etc.May there be defective in this explanation at present clinical IVF nutrient solution commonly used, that is to say that we not yet have optimal IVF nutrient solution so far.Wherein chief reason is not yet to have reliable and effective means to monitor the quality of IVF nutrient solution.
Although microarray (Microarray) analysis provides strong instrument to analyze the expression of thousands of genes 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 the single IVF case of the mankind and analyze, and in other words, Microarray can not detect as the mankind means of IVF embryo's genetic expression at present.Another kind of important situation is that different genes is very large at ovum and the expression-form difference of each pre-implantation embryos phase, especially when low by the abundance of rna transcription of cls gene, and its transcripton life-span is shorter again, and this specific character may be in the crucial effect of having regulated 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 RT-PCR of improvement, this is that a kind of analyzing gene that effectively is used for is in the method for individual cells and the expression of single implantation pre-embryo.The Real Time RT-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 very widely using value is 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 finishing in total RNA pond, be unlikely to cover the important information of the transcripton of those relatively stable expression.
To achieve the object of the present invention, the gene expression method of the single implantation pre-embryo of Real Time RT-PCR analysis IVF of the present invention's foundation 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 simultaneously amplification negative control and positive control;
Adopt the expression amount of the method testing goal gene of relative quantification; And
Carry out statistical analysis.
The invention allows for the reverse transcription reaction system in the gene expression method that utilizes Real Time RT-PCR to analyze the single implantation pre-embryo of IVF, be specially template 1.3 μ l, ThermoScript II 2.5U, random primer 2.5 μ M, dNTPs 0.5 μ M, Mg++4mM, RNase inhibitor 0.2U, 10x PCR damping fluid 0.4 μ l adds Milli Q ultrapure water to final volume 4 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 by ultra-fine glass pipette and single embryonic cell is made gene expression analysis to assess this case IVF embryo quality, determine the biological indicator of IVF pre-implantation embryos.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 determine from now on that for us new IVF and the condition of external embryo culture nutrient solution provide an important monitoring means again.
Description of drawings
By the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become apparent.Wherein:
The immunofluorescence technique and the Western blot that Figure 1 shows that a specific embodiment of the present invention analyze BCL2 and the expression of FOS in Mouse emibryo;
The Real Time RT-PCR that Figure 2 shows that a specific embodiment of the present invention is analyzed Bcl2 and the expression of Fos gene in 30 the total RNA of embryo ponds;
Figure 3 shows that Real Time RT-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;
Figure 4 shows that improvement Real Time RT-PCR of the present invention is used for analyzing gene at the step schematic diagram of the method for individual cells and the expression of single implantation pre-embryo.
Embodiment
As shown in Figure 4, according to purpose of the present invention, the present invention utilizes a kind of Real Time RT-PCR analyzing gene of improvement at the gene expression method of individual cells and single implantation pre-embryo, carry out the quality monitoring of IVF embryo medium with this, the described 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, by ultra-fine glass pipette with single zygote or pre-implantation embryos or embryonic cell, total volume is 0.1 μ L, transfer in the middle of the RNA extract of 0.9 μ L, described extract is to add 0.1IU RNA enzyme inhibitors in 1 * PCR damping fluid, collects whole RNA after processing through liquid nitrogen.
The RNA of S2, collection is further purified with RQ 1RNase-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 ℃ lower 10 minutes, add 0.1 μ L stop buffer, 95 ℃ lower 1 minute.Through the RNA of this purifying can be directly used in reverse transcription or be stored in subzero 80 ℃ for subsequent use, the longest deposit to 6 months.
S3, carry out reverse transcription reaction, its reaction system is as follows:
Add the Milli Q water of special processing to final volume 4 μ l
Wherein, the reverse transcription reaction condition is as follows:
S4, under this condition, carry out simultaneously reverse transcription negative control (not adding ThermoScript II or RNA template) and positive control (take histocyte RNA as template);
S5, reverse transcription reaction product carry out real-time quantitative PCR, and the PCR reaction system is as follows:
S6, using-system cell RNA are optimized the PCR condition, and wherein the condition of different genes primer optimization is differently (to comprise Mg
++And primer concentration, temperature of fusion, annealing temperature etc.).
PCR condition commonly used among the present invention is as follows:
S7, under this condition, amplify simultaneously amplification negative control (not adding ThermoScript II or RNA template) and positive control (comprising the Housekeeper gene, such as Actb, with the expression of goal gene in known histocyte)
S8, calculate the threshold difference (Δ Ct) of testing goal gene and Housekeeper gene in the histocyte, and the threshold difference (Δ Ct) of testing goal gene and Housekeeper gene among the embryo, relative quantification 2 adopted
-Δ Δ ctThe expression amount of method testing goal gene.
S9, statistical analysis.
According to above-mentioned method, a typical example of the present invention is that Study Mouse proto-oncogene BCL2 and FOS are in the expression of pre-implantation embryos.BCL2 and FOS are the genes of short cell survival, and Mouse Eggs and zygote are expressed 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.By Single Embryo phase embryo is implemented the real-time quantitative RT-PCR analysis, only approximately two cell stages of 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, therefore the scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention namely openly in the scope.
Claims (2)
1. a reverse transcription PCR is analyzed the reverse transcription reaction system in the gene expression method of IVF Single Embryo, and described 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 simultaneously amplification negative control and positive control;
Adopt the expression amount of the method testing goal gene of relative quantification; And
Carry out statistical analysis;
Wherein, the reaction system of described reverse transcription reaction is:
Template 1.3 μ l, ThermoScript II 2.5U, random primer 2.5 μ M, dNTPs0.5 μ M, Mg++4mM, RNase inhibitor 0.2U, 10xPCR damping fluid 0.4 μ l adds Milli Q ultrapure water to final volume 4 μ l.
2. reverse transcription PCR as claimed in claim 1 is analyzed reverse transcription reaction system in the gene expression method of IVF Single Embryo, and the reaction conditions of wherein said reverse transcription reaction is:
25 ℃ lower 10 minutes; Then 42 ℃ lower 2 minutes; Then 99 ℃ of lower 2 minutes and 4 ℃ are lower 10 minutes.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (3)
| Title |
|---|
| ZAYD A.ELDADAH ET AL.: "Marfan syndrome as a paradigm for transcript-targeted preimplantation diagnosis of heterozygous mutations", 《NATURE MEDICINE》, 31 August 1995 (1995-08-31) * |
| 张莹等: "人类卵母细胞及植入前胚胎中母性效应基因materm RNA的表达", 《现代妇产科进展》, 31 May 2009 (2009-05-31), pages 322 - 1 * |
| 邵根宝等: "小鼠植入前胚胎MuERV-I基因表达特征研究", 《南京农业大学学报》, 31 December 2007 (2007-12-31), pages 88 - 1 * |
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Application publication date: 20130109 |