CN102864219A - Method for carrying out high-flux gene expression profile detection with multiple PCR (polymerase chain reaction) matrix method - Google Patents
Method for carrying out high-flux gene expression profile detection with multiple PCR (polymerase chain reaction) matrix method Download PDFInfo
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Abstract
The invention discloses a method for carrying out high-flux gene expression profile detection with a multiple PCR (polymerase chain reaction) matrix method, which comprises multiple PCR primer design, multiple PCR matrix amplification, gel electrophoresis, image acquisition, gene expression profile quantification and clustering analysis. With the method disclosed by the invention, multiple PCR amplification and data analysis can be simultaneously carried out in PCR sample adding systems, such as a 25-hole plate, a 96-hole plate, a 384-hole plate, a 1536-hole plate and the like. The method has the advantages of large scale, high flux, high reliability and wide application prospect, and is hopeful to become one of the most important technical means for detecting the gene expression level and the high flux of nucleic acid.
Description
Technical field
The invention belongs to the multiplex PCR matrix detection method in biological technical field and the molecular diagnosis field, particularly relate to multiplex PCR matrix detection method and the application in the high-throughput expression profiling thereof of a kind of PCR of collection design of primers, multiplex PCR matrix, gel electrophoresis and IMAQ, gene expression profile quantification and cluster analysis.
Background technology
Along with the arrival in genome epoch, gene expression regulation becomes the research emphasis in molecular biology and molecular diagnostics field.Gene expression regulation detection method commonly used mainly contains at present: (Mukherjee et al.Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania.Nucleic Acids Res.2011, the doi:10.1093/nar/gkr494 such as Southern blot, gene chip, real-time quantitative PCR, extensive gene sequencing and multiplex PCR; Osamu et al.Multiplex cDNA quantification method that facilitates the standardization of gene expression data.2011,39 (10): e70.doi:10.1093/nar/gkr138.Khan et al.Quantitative analysis of six gene products as candidate markers of early placental villi development in the human.Indian J Physiol Pharmacol.2010,54 (4): 299-308; Hua et al.Genomic profile of Toll-like receptor pathways in traumatically brain-injured mice:effect of exogenous progesterone.J Neuroinflammation.2011, doi:10.1186/1742-2094-8-42).Wherein, significant to microorganism classification evaluation etc. based on the variation of multiple PCR technique mass-producing high flux screening expression of target gene level.Although traditional can realize the detection of gene expression dose based on technology such as gene chip, real-time quantitative PCR and extensive gene sequencing, because they exist false positive rate height, poor specificity, cost height and usually need reverse transcription PCR (RT-PCR) technology to verify further etc. that shortcoming makes its large-scale application be subject to certain limitation.Simultaneously; the PCR array technique that occurs in recent years is then take Real-time quantitative PCR as core and in conjunction with array technique (Zhang Yanchun; appoint Changhong; Gao Yan; Qu Wubin; Zhang Chenggang. the application of real-time quantitative PCR array technique in the mass-producing gene expression detection. institute of Military Medical Science Institute periodical; 2010; 34 (6): 589-591); can be used in the gene expression atlas of detection signal transduction process, disease-related path etc.; have certain advantage aspect the mass-producing gene expression detection, but but do not possessing high-throughout characteristics.
Summary of the invention
Be engaged in for a long time the multi-disciplinary research of PCR design of primers and assessment, gel electrophoresis and IMAQ analysis, gene expression profile quantification and cluster analysis based on the contriver, the object of the invention is to propose a kind of multiplex PCR matrix method and carried out the method for high-throughput expression profiling, be intended on the multiplex PCR basis associate(d) matrix technology and screen more on a large scale target gene, and then carry out gene expression dose quantification and cluster analysis, to obtain the expression of target gene spectrum signature.
The invention provides and a kind ofly carry out the method for high-throughput expression profiling with the multiplex PCR matrix method, comprise multiple PCR primer design, the amplification of multiplex PCR matrix, gel electrophoresis and IMAQ and gene expression profile quantification and cluster analysis.
Described multiplex PCR matrix method detects and may further comprise the steps:
1) high degree of specificity design of primers, assessment and synthetic;
2) template preparation: extraction and the reverse transcription of total RNA;
3) multiplex PCR matrix reaction system application of sample;
4) multi-PRC reaction;
5) gel electrophoresis of multiplex PCR amplified production separates;
6) gel electrophoresis images collection and pre-treatment;
7) gene expression profile quantitative analysis;
8) gene expression profile cluster analysis obtains the target gene with significant difference.
Multiple PCR primer in the described step 1) comprises: multiple PCR primer and gene specific reverse transcription primer; Multiple PCR primer is used for amplification and the evaluation that many groups have potential dependency expression of target gene spectrum, can be according to genome or the mRNA sequences Design multiple PCR primer of target gene; Gene specific reverse transcription primer is used for amplification and the evaluation of low abundance target gene, can be according to genome or the mRNA sequences Design gene specific reverse transcription primer of low abundance gene.
Described design of primers and assess available following software: MPprimer, MFEprimer2.0, Primer3.0, PrimerPremier 6.0, Oligo7.35 and DNAman are preferably MPprimer and MFEprimer2.0.
Described step 2) among the preparation method of template, at first extracts total RNA of biological cell in, then use based on the conventional reverse transcription method of universal primer or based on gene-specific primer with two step reverse transcription methods of universal primer total RNA reverse transcription to be become cDNA; Described reverse transcription method is preferably two step reverse transcription methods based on gene-specific primer and universal primer.
Sample adding system in the described step 3) can for: automatic sample-adding system and manual sample adding system, be preferably automatic sample-adding system.
Gel electrophoresis in the described step 5) can be the conventional electrophoresis such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis and pulsed field gel electrophoresis.
Gel electrophoresis images acquisition system in the described step 6) can be present commercially available various gel images acquisition systems, different amplified productions for multiplex PCR, at first adopt gel electrophoresis to separate, then gather image and combining image process software with the gel imaging instrument and carry out image pre-treatment (comprise picture delete with the adjusting of lightness etc.), carry software with the gel imaging instrument at last different gene dosages is carried out the gray-scale value analysis.
Gene expression profile quantitative analysis in the described step 7) can be the relative quantification method of internal standard gene, capillary electrophoresis or fluorescent probe absolute quantitation method etc., is preferably relative quantification method and the capillary electrophoresis absolute quantitation method of internal standard gene; The relative quantification method of described internal standard gene is: get ratio to define the relative variation tendency of gene expression profile according to the goal gene of the acquisition of the gel electrophoresis images acquisition system in the step 6) and the integral optical density value of internal standard gene; The absolute quantitation of described capillary electrophoresis is: the multiplex PCR amplified production is directly carried out capillary electrophoresis separation, then binding fluorescent dyes carries out the expression level of real-time testing goal gene, utilizes at last quantitative Marker to realize the absolute quantitation of destination gene expression level.
Gene expression profile clustering method in the described step 8) is: for the gene expression profile quantitative analysis result in the step 7), adopt bioinformatics technique that different genes is carried out cluster analysis, the concrete analysis process is as follows:
1. the gene expression profile quantitative analysis result with step 7) forms data matrix, whenever classify a treatment group as, then gene of every behavior all carries out further stdn to gene (every delegation) and sample (each row), and namely each value is divided by the standard deviation of place row or column;
2. the matrix after the 1. stdn is carried out without the supervision clustering analysis: use hierarchical cluster method in the known R software, gene and treatment group are carried out cluster analysis simultaneously;
3. the matrix after the 1. stdn is carried out the significant difference statistical study: utilize SAM algorithm in the R software that the matrix after the 1. stdn is analyzed, and utilize the q-value(quality factor in the analytical results) significant difference of gene is carried out false positive control, quality factor is defined as the significant difference gene less than 5%.
Above-described detection method is for detection of the express spectra of 33 Globin genes in the Caenorhabditis elegans, and the multiple PCR primer sequence that designs in the described step 1) is shown in sequence 1-76 in the sequence table; Described step 2) template in is Caenorhabditis elegans mRNA; Multiplex PCR matrix reaction system in the described step 3) can be: ExTaq enzyme 0.125 μ l(5U/ μ l), 10 * ExTaq Buffer2.5 μ l, dNTP Mixture2 μ l, template (cDNA concentration 300 μ g/mL) 1 μ l, multiple PCR primer 1.0 μ l(upstream primer concentration 10pmol/L, downstream primer concentration 10pmol/L), ddH2O16.375 μ l; Multi-PRC reaction condition in the described step 4) is: 95 ℃ of 5min of elder generation; Then 95 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of stopped reaction.
Described detection method can be carried out the detection of multiplex PCR matrix for gene expression dose and the dna content of any amount.
Described detection method can be carried out multiplex PCR amplification and data analysis simultaneously in the PCR application of sample systems such as 25 orifice plates, 96 orifice plates, 384 orifice plates, 1536 orifice plates.
The present inventor is based on studying for a long period of time in this field, multiple PCR technique and PCR matrix detection technique are combined, above multiplex PCR matrix detection technique has been proposed, be intended to carry out on the multiplex PCR basis more on a large scale screening-gene expression level of associate(d) matrix technology, and according to the height of gene expression dose it is carried out cluster, to obtain the expression of target gene spectrum signature.This technology not only overcome tradition based on technology for detection gene expression dose methods such as gene chip, real-time quantitative PCR and extensive gene sequencing in the deficiency aspect the reliability, but but also mass-producing, high-throughput, obtain the express spectra of target gene in high confidence.
The invention provides a kind of multiplex PCR matrix detection method and utilize this detection method to carry out the method that high-throughput nucleic acid detects; be the multiple PCR primer design integrated with the difference of existing multi-PCR detection method; the multiplex PCR matrix detects; gel electrophoresis and IMAQ; the technology such as gene expression profile quantification and cluster analysis; realized the multiple PCR primer design; the mass-producing gene test; gene expression profile quantification and cluster analysis; but has mass-producing; high-throughput; the advantages such as high confidence level; and the prospect that is widely used is expected to one of important technical that becomes the high-throughput nucleic acid detection.Report multiple PCR technique and PCR Matrix Technology are arranged respectively at present, but but do not develop the multiplex PCR Matrix Technology.For present multiple PCR technique and PCR Matrix Technology; the present invention is integrated from multiple PCR primer design and specificity assessment, multiplex PCR matrix-style detect, gene expression profile quantification and the cluster split is integrated, the novel multiple PCR system that can carry out simultaneously multiplex PCR amplification and data analysis the PCR application of sample systems such as 25 orifice plates, 96 orifice plates, 384 orifice plates, 1536 orifice plates, have the advantages such as large-scale degree height, false positive rate are low.By this multiplex PCR matrix detection method, not only can realize the quantitative analysis of gene expression profile, and can realize the cluster analysis of gene expression profile, and then between functional gene mutually the research of regulation relationship the important technology reference is provided.
Detection method of the present invention partly is comprised of PCR design of primers, multiplex PCR matrix, gel electrophoresis and IMAQ, gene expression profile quantification and cluster analysis etc., has that false positive rate is low, the large-scale degree high, and prospect is widely used.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the general frame figure of multiplex PCR matrix detection method of the present invention
The schema of Fig. 2 for multiplex PCR matrix detection method of the present invention the expression of 33 Globin genes of Caenorhabditis elegans variation being detected
The virtual electrophorogram of Fig. 3 for multiplex PCR matrix detection method and gene-specific primer the expression variation of 33 Globin genes of Caenorhabditis elegans being detected
Fig. 4 is that 1.5% agarose gel electrophoresis of interior 33 the Globin gene multiplex PCR amplified productions of Caenorhabditis elegans body in the multiplex PCR matrix detection method of the present invention detects collection of illustrative plates
Fig. 5 is for identifying result with calculation of parameter with image analysis technology to the nucleic acid band in the above-mentioned agarose gel electrophoretogram
Fig. 6 is with carrying out cluster analysis result without supervising the qualification result of hierarchical cluster method to 33 Globin genes in the Caenorhabditis elegans body
Fig. 7 is for to carry out the variance analysis result with the SAM algorithm to 33 Globin gene identification results in the Caenorhabditis elegans body
Embodiment
The invention provides a kind of multiplex PCR matrix detection method for the high-throughput expression profiling, this detection method comprises multiple PCR primer design, the amplification of multiplex PCR matrix, gel electrophoresis and IMAQ and gene expression profile quantification and cluster analysis.
Specifically, described multiplex PCR matrix detection method can may further comprise the steps:
1) high degree of specificity design of primers, assessment and synthetic;
2) template preparation: extraction and the reverse transcription of total RNA;
3) multiplex PCR matrix reaction system application of sample;
4) multi-PRC reaction;
5) gel electrophoresis of multiplex PCR amplified production separates;
6) gel electrophoresis images collection and pre-treatment;
7) gene expression profile quantitative analysis;
8) gene expression profile cluster analysis.
In above-mentioned multiplex PCR matrix detection method, the multiple PCR primer in the described step 1) comprises: conventional multiple PCR primer and gene specific reverse transcription primer; Conventional multiple PCR primer is used for amplification and the evaluation that many groups have potential dependency expression of target gene spectrum, can be according to genome or the mRNA sequences Design multiple PCR primer of target gene; Gene specific reverse transcription primer is used for amplification and the evaluation of low abundance target gene, can be according to genome or the mRNA sequences Design gene specific reverse transcription primer of low abundance gene.
Described design of primers and assess available following software: MPprimer, MFEprimer2.0, Primer3.0, PrimerPremier 6.0, Oligo7.35 and DNAman are preferably MPprimer and MFEprimer2.0.
Described step 2) among the preparation method of template, at first extracts total RNA of biological cell in, then use based on the conventional reverse transcription method of universal primer or based on gene-specific primer with two step reverse transcription methods of universal primer total RNA reverse transcription to be become cDNA; Described reverse transcription method is preferably two step reverse transcription methods based on gene-specific primer and universal primer.
Sample adding system in the described step 3) can for: automatic sample-adding system and manual sample adding system, be preferably automatic sample-adding system.
Gel electrophoresis in the described step 4) can be the conventional electrophoresis such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis and pulsed field gel electrophoresis.
Gel electrophoresis images acquisition system in the described step 5) can be present commercially available various gel images acquisition systems.Different amplified productions for multiplex PCR, at first adopt gel electrophoresis to separate, then gather image and combining image process software with the gel imaging instrument and carry out image pre-treatment (comprise picture delete with the adjusting of lightness etc.), carry software with the gel imaging instrument at last different gene dosages is carried out the gray-scale value analysis.
Gel electrophoresis images acquisition system in the described step 6) can be present commercially available various gel images acquisition systems, different amplified productions for multiplex PCR, at first adopt gel electrophoresis to separate, then gather image and combining image process software with the gel imaging instrument and carry out image pre-treatment (comprise picture delete with the adjusting of lightness etc.), carry software with the gel imaging instrument at last different gene dosages is carried out the gray-scale value analysis.
Gene expression profile quantitative analysis in the described step 7) can be the relative quantification method of internal standard gene, capillary electrophoresis or fluorescent probe absolute quantitation method etc., is preferably relative quantification method and the capillary electrophoresis absolute quantitation method of internal standard gene; The relative quantification method of described internal standard gene is: get ratio to define the relative variation tendency of gene expression profile according to the goal gene of the acquisition of the gel electrophoresis images acquisition system in the step 6) and the integral optical density value of internal standard gene; The absolute quantitation of described capillary electrophoresis is: the multiplex PCR amplified production is directly carried out capillary electrophoresis separation, then binding fluorescent dyes carries out the expression level of real-time testing goal gene, utilizes at last quantitative Marker to realize the absolute quantitation of destination gene expression level.
Gene expression profile clustering method in the described step 8) is: for the gene expression profile quantitative analysis result in the step 7), adopt bioinformatics technique that different genes is carried out cluster analysis, the concrete analysis process is as follows:
1. the gene expression profile quantitative analysis result with step 7) forms data matrix, whenever classify a treatment group as, then gene of every behavior all carries out further stdn to gene (every delegation) and sample (each row), and namely each value is divided by the standard deviation of place row or column;
2. the matrix after the 1. stdn is carried out without the supervision clustering analysis: use hierarchical cluster method in the known R software, gene and treatment group are carried out cluster analysis simultaneously;
3. the matrix after the 1. stdn is carried out the significant difference statistical study: utilize SAM algorithm in the R software that the matrix after the 1. stdn is analyzed, and utilize the q-value(quality factor in the analytical results) significant difference of gene is carried out false positive control, quality factor is defined as the significant difference gene less than 5%.
In the above step, step 7) in conjunction with the integral optical density value of reference gene other each band is carried out stdn, increased the comparability of different experiments group amplifying nucleic acid electrophoretic band signal value, improved the quantitative effect of whole method system; The larger characteristics of noise were carried out further stdn to data when 1. step detected for the multiplex PCR matrix especially, had improved specifically the signal to noise ratio that genetic expression detects; 2. and 3. step will be inherited for the method for gene chip data analysis in the world, brought into play the visual etc. of high throughput method in the multiplex PCR matrix, can be intuitively, clearly the expression of gene changed display, and adopt strict statistical method to carry out difference test.These steps all are different from classical PCR experimental technique significantly, are the peculiar data analysing methods of multiplex PCR matrix method.
In the following embodiments, the expression that detects 33 Globin genes in the Caenorhabditis elegans with multiplex PCR matrix detection method of the present invention changes, and the multiple PCR primer sequence that designs in the described step 1) is shown in sequence 1-76 in the sequence table; Described step 2) template in is Caenorhabditis elegans mRNA; Multiplex PCR matrix reaction system in the described step 3) can be: ExTaq enzyme 0.125 μ l(5U/ μ l), 10 * ExTaq Buffer2.5 μ l, dNTP Mixture2 μ l, template (cDNA concentration 300 μ g/mL) 1 μ l, multiple PCR primer 1.0 μ l(upstream primer concentration 10pmol/L, downstream primer concentration 10pmol/L), ddH
2O16.375 μ l; Multi-PRC reaction condition in the described step 4) is: 95 ℃ of 5min of elder generation; Then 95 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of stopped reaction.The matrix quantity of this embodiment is 8 groups, namely is divided into 8 pipes and carries out multiplex PCR amplification, wherein used respectively the heavy PCR of 4-6 to describe in every group, consisted of one contain 8 groups of amplification systems, contain the heavy multiplex PCR matrix amplification system of 4-6.Similarly, the present invention also can carry out multiplex PCR amplification and data analysis simultaneously in the PCR application of sample systems such as 25 orifice plates, 96 orifice plates, 384 orifice plates, 1536 orifice plates.
Method therefor is ordinary method if no special instructions among the following embodiment.
As shown in Figure 1, multiplex PCR matrix detection method of the present invention detects the express spectra of goal gene, be the pre-treatment (comprising conservative Analysis and design of primers regional analysis etc.) before at first obtaining gene order for goal gene and carrying out the multiple PCR primer design, and then adopt design of primers and specificity assessment software to carry out the multiple PCR primer design for the goal gene sequence; And then the primer for design dilutes, prepares template and multiplex PCR increases to obtain goal gene; Then adopt gel electrophoresis to separate, gather image and pre-treatment for the multiplex PCR amplified production; Carry out quantification and cluster analysis for the genetic expression qualification result at last.Specifically, detect with the express spectra of multiplex PCR matrix detection method of the present invention to 33 Globin genes of Caenorhabditis elegans, as shown in Figure 2, concrete grammar may further comprise the steps:
1) high degree of specificity design of primers, assessment and synthetic
For 33 Globin gene informations of Caenorhabditis elegans, take act-1 as reference gene, download the genes involved sequence from ncbi database respectively, and then adopt multiple PCR primer design software MPprimer (http://biocompute.bmi.ac.cn/MPprimer/) to carry out the multiple PCR primer design, the multiple PCR primer sequence is as shown in table 1, and (table 1 altogether primer has 76,38 pairs), and (virtual electrophorogram is as shown in Figure 3 (among the figure to adopt primer specificity assessment software MFEprimer2.0 (http://biocompute.bmi.ac.cn/MFEprimer/) to carry out specificity assessment and virtual analysis, laterally 1-34 represents different tracks successively, swimming lane 1-33 (from left to right) is the substance virtual result, swimming lane 34 (the rightest one) is 33 heavy virtual result, wherein white ribbon represents the different IPs acid fragment), the primer with design send handsome company directly synthetic for subsequent use at last.By above-mentioned design of primers and specificity appraisal procedure, can obtain according to experiment purpose the multiplex PCR amplimer of any goal gene.
Table 1 is used for the express spectra to 33 Globin genes of Caenorhabditis elegans
Carry out the multiple PCR primer sequence that the multiplex PCR matrix detects
2) template preparation
At first the normal strain of Caenorhabditis elegans is cultivated the L2 phase, then adopt 0.5g/L concentration S-WAT to prepare the Caenorhabditis elegans hypoxia model, and respectively at processing rear 8 hours, 10 hours, collected nematode in 12 hours and 14 hours, then utilize the total RNA extraction reagent box to extract total RNA of nematode, concrete grammar is: be transferred in the tissue homogenizer after Caenorhabditis elegans is adopted M9 buffer solution for cleaning three times, then add 350 μ l and contain the TRK lysis buffer (adding 20 μ l beta-mercaptoethanols among the 1mL TRK Lysis Buffer) of beta-mercaptoethanol and be ground to evenly; To grind uniform liquid and be drawn in the 1.5mL EP pipe, the centrifugal 5min of 14000r/min room temperature gets in the new 1.5mLEP pipe of supernatant to; In the EP pipe, add 70% ethanol that 350 μ l process with DEPC, the piping and druming mixing; The liquid that mixes is added on the centrifugal column the centrifugal 1.5min of 10000r/min room temperature; In centrifugal column, add 300 μ l RNA WashBuffer I, the centrifugal 1.5min of 12000r/min room temperature; DNase I digestion process (73.5 μ l DNase I Digestion Buffer and 1.5 μ l RNase-free DNase I), room temperature leaves standstill 15min; Add 500 μ lRNA Wash Buffer I, the centrifugal 1.5min of 10000r/min room temperature; The RNA WashBuffer II 500 μ l that adding was diluted with dehydrated alcohol, the centrifugal 1.5min of 10000r/min room temperature; Again add 500 μ l RNA Wash Buffer II, the centrifugal 1.5min of 10000r/min room temperature; The collection tube that renews, 120000r/min room temperature sky gets rid of 2.5min; Pillar is inserted in the new 1.5mL centrifuge tube, adds 40 μ l DEPC water, the centrifugal 1.5min of 10000r/min room temperature collects RNA to centrifuge tube; Take out 1 μ l RNA and dilute 100 times, utilize UV spectrophotometer measuring its at the light absorption value of 260nm, 280nm and calculate its purity and concentration, all the other are put in-80 ℃ of preservations.
At last with total RNA of said extracted to specifications behind the application of sample reverse transcription become cDNA with as the multiplex PCR amplification template, concrete grammar is: according to following application of sample system application of sample: total RNA1 μ g+10pmol/L oligo d (T)
121 μ l+RNase Free water polishing to 12 μ l, rapidly ice bath cooling behind 65 ℃ of sex change 5min, then add 4 μ l5 * RT buffer, 2 μ l dNTP, 1 μ l RNase Inhibitor and 1 μ l Rever TraAce carry out reverse transcription (step is: 30 ℃ * 10min → 42 ℃ * 30min → 85 ℃ * 5min → 4 ℃ stop).
3) multiplex PCR matrix reaction system application of sample
Adopt automatic sample-adding system to carry out multiplex PCR matrix reaction system application of sample according to following application of sample amount: ExTaq enzyme 0.125 μ l(5U/ μ l), 10 * ExTaq Buffer2.5 μ l, dNTP Mixture2 μ l, template (cDNA concentration 300 μ g/mL) 1 μ l, multiple PCR primer 1.0 μ l(upstream primer concentration 10pmol/L, downstream primer concentration 10pmol/L), ddH
2O 16.375 μ l.
4) multi-PRC reaction
Above-mentioned application of sample system is put into the PCR detector, carry out multiplex PCR amplification according to following multi-PRC reaction condition and be: first 95 ℃ of 5min; Then 95 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of stopped reaction.
5) gel electrophoresis of multiplex PCR amplified production separates
The agarose gel electrophoresis of above-mentioned multiplex PCR amplified production employing 1.5% is separated the multiplex PCR amplified production, obtain agarose gel electrophoretogram, as shown in Figure 4.
6) gel electrophoresis images collection and pre-treatment
Adopt gel imaging instrument (model C hampGel for above-mentioned agarose gel electrophoresis separation electrophoresis figure (Fig. 4)
TM5000, available from Saizhi Chuangye Science and Technology Co., Ltd., Beijing) the collection image, and then in conjunction with Photoshop, the image processing softwares such as Picture manager carry out image pre-treatment (comprise picture delete adjusting with lightness etc.) to agarose gel electrophoretogram, the Lane 1D software that carries with the gel imaging instrument at last carries out integral optical density value (the Integrated Optical Density that gray-scale value analysis obtains each band to different gene bands, IOD, this IOD value and gene expression amount positive correlation), thus raw data provided for quantification and the cluster analysis of follow-up gene expression profile.Lane 1D software for the analytical results of 33 Globin genes of Caenorhabditis elegans as shown in Figure 5, wherein, horizontal swimming lane 1-8 represents respectively normal oxygen cultivation 8 hours, anoxic cultivation 8 hours, often oxygen cultivation 10 hours, anoxic cultivation 10 hours, often oxygen cultivation 12 hours, anoxic cultivation 12 hours, often oxygen cultivation 14 hours, anoxic cultivation 14 hours, longitudinal band 1-6 represents respectively Globin-30, Globin-29, Globin-26, Globin-27, Actin-1, Globin-28, and Marker is DL2000 DNA Marker.
7) gene expression profile quantitative analysis
The gene expression profile quantitative analysis can adopt two kinds of methods:
It is 1. based on internal standard gene relative quantification method: utilize internal standard gene to define the relative expression quantity of goal gene, with the integral optical density value of each gene obtained above integral optical density value divided by its internal standard gene, the ratio of gained the results are shown in Table 2 as relative expression quantity.Along with the increase of anoxic time, the up-regulated of glb-5, but descended to some extent in 14 hours in anoxic; The down-regulated expression of glb-8, anoxic group decline level was maximum in 10 hours; The expression level of glb-11 raises, but 14 hours anoxic groups descend obviously; The expression level of glb-13 increases first rear minimizing, illustrates anoxic has been produced adaptability, and prompting glb-13 may be the hypoxia regulatory gene; Glb-14, the downward modulation of 22 expression levels; The glb-29 up-regulated.
33 Globin genes of C. Elegans Automatic Screening that table 2 obtains based on the multiplex PCR Matrix Technology
Expression level after the normal oxygen of different time and anaerobic treatment
Unit: relative expression quantity
Its 2. absolute quantitation method based on capillary electrophoresis: with above-mentioned steps 1) the multiplex PCR matrix amplified production that-4) obtains can directly adopt capillary electrophoresis to separate, and mark Marker carries out the absolute quantification analysis of gene expression amount in obtaining to utilize behind the peak figure of each gene band.
8) gene expression profile cluster analysis
For the gene expression profile quantitative analysis result in the step 7), adopt bioinformatics technique that different genes is carried out cluster analysis, the concrete analysis process is as follows:
1. the gene expression amount in the step 7) (relative expression quantity is as shown in table 2) is formed data matrix, whenever classify a treatment group as, gene of every behavior, then gene (every delegation) and sample (each row) are all carried out further stdn, namely each value is divided by the standard deviation of place row or column;
2. the matrix after the 1. stdn is carried out without the supervision clustering analysis: use hierarchical cluster method in the known R software, gene and treatment group are carried out cluster simultaneously, the result laterally represents each experiment grouping as shown in Figure 6, vertically represents the different genes that multiplex PCR detects; Green (among Fig. 6 than shallow portion) expression cance high-expression gene, the low expressing gene of red (dividing than the deep among Fig. 6) expression, black (deepest part among Fig. 6) represents medium expressing gene, can find out that different detection genes (X-coordinate is listed) (ordinate zou is listed) under different experiment conditions shows collaborative or different expression changing patteries, shows that present method can detect the genetic expression of each quasi-mode under the different experimental conditions effectively simultaneously;
3. the matrix after the 1. stdn is carried out the significant difference statistical study: utilize (the Significance Analysis of Microarrays of SAM in the R software, the microarray significance analysis, can be used for the restricted check of the gene differential expression on the statistical significance) algorithm analyzes (the variance analysis result to 33 Globin gene expression profiles in the Caenorhabditis elegans body is as shown in table 3) to the matrix after the 1. stdn, and utilize the q-value(quality factor in the analytical results, claim again False Discovery Rate, as molecule take the false positive event number, calculate take false positive and true positive events quantity as denominator) significant difference of gene is carried out false positive control, quality factor is defined as significant difference gene (to the variance analysis of 33 Globin gene identification results in the Caenorhabditis elegans body as shown in Figure 7) less than 5%.
The variance analysis result of 33 Globin gene expression profiles in the table 3 Caenorhabditis elegans body
Can find out in the Caenorhabditis elegans body 33 Globin genes 29 positive regulating genes and 4 negative regulator genes are arranged from table 3 and Fig. 7, it is remarkable positive regulating gene that 3 genes (glb-8, glb-11, glb-5 gene) are wherein arranged, shown in grayish square points among Fig. 7, the observation score value is higher than the threshold value of expecting score value, show that these three genes are remarkable up-regulated gene, q-value is 0 simultaneously.
Present embodiment has obtained the expression amount of 33 genes of C. Elegans Automatic Screening globin under different experiment conditions by above step, has obtained three genes of significantly just regulating and control, and can determine that it is the relevant target gene of anoxic.
More than used the detection of expression of 33 genes of C. Elegans Automatic Screening globin as embodiment, only as an example application, not as a limitation of the invention.The inventive method can be used for a large amount of arbitrarily detections of gene, it is the advantage place of the method, can be used in theory detecting the expression level of whole Human genomes or groups of people's genoid and other species genes, and contain the nucleic acid content in the complex sample of identical source or different sources.
Claims (13)
1. one kind is carried out the method for high-throughput expression profiling with the multiplex PCR matrix method, comprises multiple PCR primer design, the amplification of multiplex PCR matrix, gel electrophoresis and IMAQ and gene expression profile quantification and cluster analysis.
2. detection method according to claim 1 is characterized in that, the multiplex PCR matrix method detects and may further comprise the steps:
1) high degree of specificity design of primers, assessment and synthetic;
2) template preparation: extraction and the reverse transcription of total RNA;
3) multiplex PCR matrix reaction system application of sample;
4) multi-PRC reaction;
5) gel electrophoresis of multiplex PCR amplified production separates;
6) gel electrophoresis images collection and pre-treatment;
7) gene expression profile quantitative analysis;
8) gene expression profile cluster analysis obtains the target gene with significant difference.
3. detection method according to claim 2, it is characterized in that: the multiple PCR primer in the described step 1) comprises: multiple PCR primer and gene specific reverse transcription primer; Multiple PCR primer is used for amplification and the evaluation that many groups have potential dependency expression of target gene spectrum, can be according to genome or the mRNA sequences Design multiple PCR primer of target gene; Gene specific reverse transcription primer is used for amplification and the evaluation of low abundance target gene, can be according to genome or the mRNA sequences Design gene specific reverse transcription primer of low abundance gene.
4. detection method according to claim 3, it is characterized in that: described design of primers and assess available following software: MPprimer, MFEprimer2.0, Primer3.0, Primer Premier 6.0, Oligo7.35 and DNAman are preferably MPprimer and MFEprimer2.0.
5. according to the described detection method of above arbitrary claim, it is characterized in that: described step 2) among the preparation method of middle template, at first extract total RNA of biological cell, then use based on the conventional reverse transcription method of universal primer or based on gene-specific primer with two step reverse transcription methods of universal primer total RNA reverse transcription to be become cDNA; Described reverse transcription method is preferably two step reverse transcription methods based on gene-specific primer and universal primer.
6. according to the described detection method of above arbitrary claim, it is characterized in that: the sample adding system in the described step 3) can for: automatic sample-adding system and manual sample adding system, be preferably automatic sample-adding system.
7. according to the described detection method of above arbitrary claim, it is characterized in that: the gel electrophoresis in the described step 5) can be the conventional electrophoresis such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis and pulsed field gel electrophoresis.
8. according to the described detection method of above arbitrary claim, it is characterized in that: the gel electrophoresis images acquisition system in the described step 6) can be present commercially available various gel images acquisition systems, different amplified productions for multiplex PCR, at first adopt gel electrophoresis to separate, then gather image and combining image process software with the gel imaging instrument and carry out image pre-treatment (comprise picture delete with the adjusting of lightness etc.), carry software with the gel imaging instrument at last different gene dosages is carried out the gray-scale value analysis.
9. according to the described detection method of above arbitrary claim, it is characterized in that: the gene expression profile quantitative analysis in the described step 7) can be the relative quantification method of internal standard gene, capillary electrophoresis or fluorescent probe absolute quantitation method etc., is preferably relative quantification method and the capillary electrophoresis absolute quantitation method of internal standard gene;
The relative quantification method of described internal standard gene is: get ratio to define the relative variation tendency of gene expression profile according to the goal gene of the acquisition of the gel electrophoresis images acquisition system in the step 6) and the integral optical density value of internal standard gene;
The absolute quantitation of described capillary electrophoresis is: the multiplex PCR amplified production is directly carried out capillary electrophoresis separation, then binding fluorescent dyes carries out the expression level of real-time testing goal gene, utilizes at last quantitative Marker to realize the absolute quantitation of destination gene expression level.
10. according to the described detection method of above arbitrary claim, it is characterized in that: the gene expression profile clustering method in the described step 8) is: for the gene expression profile quantitative analysis result in the step 7), adopt bioinformatics technique that different genes is carried out cluster analysis, the concrete analysis process is as follows:
1. the gene expression profile quantitative analysis result with step 7) forms data matrix, whenever classify a treatment group as, then gene of every behavior all carries out further stdn to gene (every delegation) and sample (each row), and namely each value is divided by the standard deviation of place row or column;
2. the matrix after the 1. stdn is carried out without the supervision clustering analysis: use hierarchical cluster method in the known R software, gene and treatment group are carried out cluster analysis simultaneously;
3. the matrix after the 1. stdn is carried out the significant difference statistical study: utilize SAM algorithm in the R software that the matrix after the 1. stdn is analyzed, and utilize the q-value(quality factor in the analytical results) significant difference of gene is carried out false positive control, quality factor is defined as the significant difference gene less than 5%.
11. each described detection method according to claim 1-10 is characterized in that: detect the express spectra of 33 Globin genes in the Caenorhabditis elegans, the multiple PCR primer sequence that designs in the described step 1) is shown in sequence 1-76 in the sequence table; Described step 2) template in is Caenorhabditis elegans mRNA; Multiplex PCR matrix reaction system in the described step 3) can be: ExTaq enzyme 0.125 μ l(5U/ μ l), 10 * ExTaq Buffer, 2.5 μ l, dNTPMixture 2 μ l, template (cDNA concentration 300 μ g/mL) 1 μ l, multiple PCR primer 1.0 μ l(upstream primer concentration 10pmol/L, downstream primer concentration 10pmol/L), ddH
2O 16.375 μ l; Multi-PRC reaction condition in the described step 4) is: 95 ℃ of 5min of elder generation; Then 95 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 7min, 4 ℃ of stopped reaction.
12. each described detection method is characterized in that: can carry out the detection of multiplex PCR matrix for gene expression dose and the dna content of any amount according to claim 1-11.
13. each described detection method is characterized in that: can carry out simultaneously multiplex PCR amplification and data analysis in the PCR application of sample systems such as 25 orifice plates, 96 orifice plates, 384 orifice plates, 1536 orifice plates according to claim 1-12.
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