CN100577817C - Relative quantitative method for analyzing prokaryote gene expression by utilizing real time fluorescence reverse transcription PCR - Google Patents

Abstract

The invention relates to a relative quantitative method for analyzing the prokaryote gene expression through real-time fluorescence inverse transcription PCR, and belongs to the basic biology and medicine field. The invention solves the problems that the housekeeping gene expression is unstable in the residual DNA and during the bacterial growth process, and when the PCR efficiency is not high or the difference of the PCR efficiency among a plurality of gene is larger, the error of the analyzed result is larger, to cause the practical expression of the prior gene expression relative quantitative to have limitation. The invention has the analysis method that the concurrent DNA of the RNA extracted product is taken as the internal standard, and RT, and RT<-> samples are prepared, the prokaryote gene expression quantity can be calculated through the relative quantitative formula, wherein, Q means the expression quantity of the target gene, Q1 means the expression quantity of the reference gene, Delta Ct is equal to CtRT-CtRT-, Delta Ct1 is equal to CtRT-CtRT-1, E is equal to 10<[-1/slope>]-1, the CT value means the circulation number when the fluorescence signal reaches the threshold value, the RT means the inverse transcription, and RT<-> means non-inverse transcription. The invention has the advantages that the process is simple, the operation is easy, a great amount of samples can be detected once, the result is direct, the sensitivity is high, the specificity is strong, and the repeatability is good.

Description

A kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression

Technical field

The invention belongs to fundamental biological knowledge and medical field, relate to a kind of relative quantitative assay method of prokaryote gene expression, specifically be meant the relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression.

Background technology

Along with the continuous development of biological gene group and information biology and going deep into of functional genomics research, gene expression research becomes research emphasis in recent years.Prokaryotic organism are the important integral part of whole organic sphere, and are closely related with the human existence activity, and corresponding research has phased out into the exploratory study in functional genome field from structural genomics, thereby finally realize the foundation of prokaryotic organism systems biology.Research to prokaryote gene expression mainly is to analyze from the gene transcription level at present, just carries out the research of qualitative, quantitative from the RNA angle.Real time fluorescent quantitative reverse transcription PCR (the Real-time RT PCR) technology of being come by the reverse transcription PCR development all is used widely in fields such as biology, medical science, food inspection, becomes the important method of on the gene transcription level genetic expression being analyzed.

Having now utilizes Real-time RT round pcr prokaryote gene expression to be carried out in the entire operation computation process of relative quantitative assay, consider based on different experiments, processing mode to following three key components often has nothing in common with each other, and also there is certain deficiency respectively in the method for being taked:

1, all inevitably having part DNA among the RNA with any method extraction exists.Can design or the mRNA purifying by the intron of striding of PCR primer for eukaryote, eliminate the DNA influence in the RNA detection.And procaryotic mRNA does not contain intron and polyA structure, generally be to utilize DNaseI (RNase Free) that its DNA is handled, promptly do not wait, remove DNase I enzyme through high temperature to DNase I deactivation or by phenol-chloroform extracting again at 37 ℃ of enzymolysis 10-30 minutes.Because the less stable of procaryotic RNA own, above-mentioned treating processes can affect greatly total amount and the quality of RNA.

2, in the RNA relative quantitative assay,, all can cause being used to extract initial thalline/cell concentration difference of RNA owing to the different treatment mode or the difference in operation of thalline/cell.If with unified the extraction to identical thalline/cell concentration of specimen material is very difficult, should selects a kind of gene of in the entire treatment process, transcribing horizontal stable initial thalline/cell concentration to be carried out homogenization and proofread and correct as interior mark.The general 16S of selection rRNA etc. are as marking in procaryotic.But 2002, people such as Vandecasteele SJ studies show that, the content of 16S rRNA is also unstable, also bigger fluctuation can occur at the different growing stage of thalline.

3, the concrete analysis algorithm of genetic expression relative quantification.Relevant algorithm has a variety of, carry out relative quantification with external standard method at first, promptly make typical curve, determine the definite template amount of goal gene and house-keeping gene (House keeping gene) according to the external standard of a series of known copy numbers, with both ratio as the net result of relative quantification.This method need be carried out the plasmid clone of extension increasing sequence, and determines copy number by detection computations, just can draw out the absolute quantitation typical curve.This relative quantitative assay process based on absolute quantitation is loaded down with trivial details, easily produces error.The kinetic model of PCR-based amplification afterwards occurs (1+E) ^{-Δ Δ Ct}Algorithm: Q _R=(1+E) ^{-Δ Δ Ct}, Q wherein _RBe the result of the relative quantification of goal gene in goal gene in the experimental group and the control group, Δ Δ Ct=(Ct _{Goal gene}-Ct _{Reference gene}) _{Experimental group}-(Ct _{Goal gene}-Ct _{Reference gene}) _{Control group}, the Ct value is meant the cycle number when fluorescent signal reaches threshold value, E is a PCR efficient.But the calculating of PCR efficient is in the past calculated by the slope meter of the absolute quantitation typical curve of known definite copy number, makes entire operation, computation process more complicated, is difficult for promoting the use of.ABI company when releasing ABI 7700 type Real-time PCR instrument, released 2 ^{-Δ Δ Ct}Method (ABI, Sequence Detector User Bulletin 2): Q _R=2 ^{-Δ Δ Ct}The efficient E of each PCR reaction is 1 in this algorithm hypothesis relative quantification, can be used for the gene transcription level is carried out roughly relative quantitative assay, when but PCR efficient difference is big between not high or several genes when PCR efficient, the resultant error that is analyzed is bigger, has certain limitation in actual applications.

Summary of the invention

The invention provides a kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression, purpose is residual in order to solve DNA, and in the thalli growth process house-keeping gene unstable expression and when PCR efficient PCR efficient difference greatly time the between not high or several genes, the resultant error that is analyzed is bigger, causes existing genetic expression relative quantification to have certain circumscribed problem in actual applications.

Utilize the relative quantification method of real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression, described analytical procedure realizes by following process: the cDNA that, utilizes detected prokaryotic organism RNA to obtain for the template reverse transcription carries out gradient dilution and draws out the doubling dilution typical curve, obtain slope slope, by PCR effectiveness formula E=10 ^[1/slope]-1 calculates Real-time PCR efficient E; Two, the extraction of procaryotic total nucleic acid: utilize phenol-chloroform method to extract and be in the logarithmic growth detected procaryotic total nucleic acid in mid-term; Three, the total nucleic acid of being extracted is prepared reverse transcription sample and non-reverse transcription sample, the preparation method is as follows: the detected prokaryotic organism total nucleic acid through the step 2 extraction of getting two equal portions; Wherein portion is carried out reverse transcription reaction obtain the reverse transcription sample; Is that 0.1% DEPC water is diluted to the concentration identical with the RT sample with another part with volumetric concentration, obtains non-reverse transcription sample; Four, reverse transcription sample and the non-reverse transcription sample that obtains in step 3 carries out Real-time PCR detection, will obtain the Ct of reverse transcription sample in the step 3 respectively _RTThe Ct of value and non-reverse transcription sample _RT-value; Five, the analysis expressed of prokaryotic gene relative quantification: specify that one group of gene expression amount Q ' passes through the relative quantification formula for the data that reference group adopts above-mentioned steps to record in the detected prokaryotic organism $Q_R=\frac{Q}{Q^{\&prime;}}=\frac{{(1+E)}^{-\mathrm{\&Delta;Ct}}-1}{{(1+E)}^{-\mathrm{\&Delta;}{\mathrm{Ct}}^{\&prime;}}-1}$ Calculate the relative expression quantity Q of prokaryotic gene _RWherein Q represents the expression amount of goal gene, the expression amount of Q ' expression reference gene, $\mathrm{\&Delta;Ct}={\mathrm{Ct}}_{\mathrm{RT}}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}},$ ${\mathrm{\&Delta;Ct}}^{\&prime;}={\mathrm{Ct}}_{\mathrm{RT}}^{\&prime;}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}}^{\&prime;},$ E=10 ^[1/slope]-1, the Ct value is meant the cycle number when fluorescent signal reaches threshold value, and RT represents reverse transcription, RT ^-Represent non-reverse transcription.

The present invention is for keeping the quality of RNA in the total nucleic acid, and all utensils and related solution are all handled through diethylpyrocarbonate (DEPC) aqueous solution of 0.1% (volume), and the total nucleic acid of extracting finally is dissolved in the DEPC water of 0.1% (volume).In step 1, detect in the total nucleic acid whether comprise DNA and RNA with agarose gel electrophoresis; Total nucleic acid is carried out the mensuration of ultraviolet light absorption value, detect the purity of nucleic acid extraction.

In conventional fluorescent quantitation reverse transcription PCR genetic expression relative quantitative assay method and (1+E) ^{-Δ Δ Ct}On the basis of algorithm, at the characteristics of RNA in the prokaryote gene expression, the present invention has set up a kind of relative quantification detection architecture in conjunction with the DNA substractive process: with in the RNA extract and deposit DNA as interior mark, the preparation RT, RT ^-Sample, and therefrom carry out the deduction (replacing original enzymolysis step) of DNA, and mark used in the content of DNA also can be used as, detect under cost and the prerequisite of time not increasing, solved simultaneously have in the genetic expression relative quantitative assay that DNA is residual, house-keeping gene unstable expression and 2 in the thalli growth process ^{-Δ Δ Ct}Algorithm can't carry out the problem of the effective evaluation of PCR efficient, and analytical procedure of the present invention can be carried out effective relative quantitative assay to expression of gene in the prokaryotic organism.

Compared with prior art, mainly being presented as of the positively effect of the present invention in prokaryote gene expression real-time fluorescent reverse transcription PCR relative quantitative assay is used:

1, the present invention need not know definite copy number, and the measuring method complexity of copy number in the actually operating, the present invention detects and reduces the cost;

2, can be residual by the DNA that analytical calculation is removed in the RNA extraction, saved the step of enzymolysis DNA, shortened detection time and cost;

3, can utilize once more detected dna content data, carry out the homogenization of biomass as mark in the DNA and proofread and correct, further reduce and detect cost;

4, guaranteeing to utilize formula on the objective and accurate basis of detected result: $Q_R=\frac{Q}{Q^{\&prime;}}=\frac{{(1+E)}^{-\mathrm{\&Delta;Ct}}-1}{{(1+E)}^{-{\mathrm{\&Delta;Ct}}^{\&prime;}}-1},$ Can carry out simple and rapid computational analysis to detected result, and practical ranges is wider.

The present invention has simple, easy to operate, the disposable advantages such as a large amount of sample detection, visual result, susceptibility height, high specificity, good reproducibility of carrying out of process.Method of the present invention can be carried out effective relative quantitative assay to expression of gene in the prokaryotic organism, and this relative quantitative assay method has stronger directive significance to the relative quantitative assay of prokaryote gene expression.

Embodiment

Embodiment one: the relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression in the present embodiment, this method can disposablely be carried out relative quantitative assay to different genes, described analytical procedure is finished by following step: the cDNA that, utilizes detected prokaryotic organism RNA to obtain for the template reverse transcription carries out gradient dilution and draws out the doubling dilution typical curve, obtain slope slope, by PCR effectiveness formula E=10 ^[1/slope]-1 calculates Real-time PCR efficient E; Two, the extraction of procaryotic total nucleic acid: utilize phenol-chloroform method to extract and be in the logarithmic growth detected procaryotic total nucleic acid in mid-term (DNA and RNA); Three, the total nucleic acid of being extracted is prepared reverse transcription sample and non-reverse transcription sample, the preparation method is as follows: get the detected prokaryotic organism total nucleic acid through the step 2 extraction of two equal portions; Wherein portion is carried out reverse transcription reaction obtain reverse transcription sample (contain cDNA and DNA, next step PCR of the two fellowship detects); Is that 0.1% DEPC water is diluted to the concentration identical with the RT sample with another part with volumetric concentration, obtains non-reverse transcription sample (only having DNA to participate in next step PCR detects); Four, reverse transcription sample and the non-reverse transcription sample that obtains in step 3 carries out Real-time PCR detection, will obtain the Ct of reverse transcription sample in the step 3 respectively _RTThe Ct of value and non-reverse transcription sample _RT-value; Five, the analysis expressed of prokaryotic gene relative quantification: specify that one group of gene expression amount Q ' passes through the relative quantification formula for the data that reference group adopts above-mentioned steps to record in the detected prokaryotic organism $Q_R=\frac{Q}{Q^{\&prime;}}=\frac{{(1+E)}^{-\mathrm{\&Delta;Ct}}-1}{{(1+E)}^{-{\mathrm{\&Delta;Ct}}^{\&prime;}}-1}$ Calculate the relative expression quantity Q of prokaryotic gene _RWherein Q represents the expression amount of goal gene, the expression amount of Q ' expression reference gene, $\mathrm{\&Delta;Ct}={\mathrm{Ct}}_{\mathrm{RT}}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}},$ ${\mathrm{\&Delta;Ct}}^{\&prime;}={\mathrm{Ct}}_{\mathrm{RT}}^{\&prime;}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}}^{\&prime;},$ E=10 ^[1/slope]-1, the Ct value is meant the cycle number when fluorescent signal reaches threshold value, and RT represents reverse transcription, RT ^-Represent non-reverse transcription.

Embodiment two: present embodiment and embodiment one are different is that slope calculations slope method in the described step 1 is as follows: cDNA is carried out 5 10 times of gradient dilutions respectively, carry out Real-time PCR then and detect; If the minimum template concentrations of dilution is 10 ¹, with the logarithmic value (lg10 of template concentrations multiple ¹～lg10 ⁵) be X-coordinate, the Ct value is an ordinate zou drawing standard curve, obtains slope slope.Other reactions steps is identical with embodiment one.

Embodiment four: present embodiment and embodiment one are different is that to extract the total nucleic acid method in the described step 2 as follows: the bacterial strain of first activating gene, bacterial isolates after will activating then is inoculated in the substratum, under suitable bacterial isolates growth temperature, cultivate logarithmic growth during mid-term, thalline is collected in the centrifugation of bacterium liquid, utilized conventional phenol-chloroform method to extract total nucleic acid again.Other reactions steps is identical with embodiment one.

Embodiment five: present embodiment and embodiment one are different is that the RT sample preparation methods of gene in the described step 3 is as follows: the step 1 of learning from else's experience method is extracted detected prokaryotic organism total nucleic acid and is utilized ExScript ^TMThe reverse transcription test kit carries out reverse transcription reaction and carries out reverse transcription reaction, and the temperature of reverse transcription is 42 ℃, and the time of reverse transcription is 15min, and the temperature of ThermoScript II inactivation is 95 ℃, and the time of ThermoScript II inactivation is 2min, obtains the RT sample of gene.Other reactions steps is identical with embodiment one.

Embodiment four: what present embodiment and embodiment one were different is to detected procaryotic RT sample and RT in the described step 4 ^-Sample carries out pcr amplification, after the PCR reaction conditions is 95 ℃ of sex change 10s, and 95 ℃ of 5s, 40 circulations of 60 ℃ of 34s amplifications will be collected fluorescent signal and be set at threshold value, at the Ct that measures the RT sample respectively when 60 ℃ of 34s _RTValue and RT ^-Sample and Ct _RT-Value.Other reactions steps is identical with embodiment one.

Embodiment six: enterotoxin A gene (sea), three kinds of gene relative quantification expression amounts of 16S rRNA, RNAIII (effector of agr regulator control system) in the method for utilizing real-time fluorescent reverse transcription PCR of the present invention in the present embodiment and the streptococcus aureus that conventional quantitative method analysis is cultivated in the NB of different glucose concn substratum, the A gene (sea), 16S rRNA, RNAIII expression amount (Q ') that with the glucose concn are the streptococcus aureus cultivated in the NB substratum of 0mmol/L respectively are reference group, the results are shown in Table 1:

Table 1

Method of the present invention compares with classical absolute quantitation method relative quantitative assay in the his-and-hers watches 1, difference less (all less than 15%), and result difference not significantly (p＞0.05), so the bright method of this law can obtain the result similar to the absolute quantitation method.Analytical procedure of the present invention can be carried out effective relative quantitative assay to expression of gene in the prokaryotic organism.

Claims (5)

1, a kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression, the step that it is characterized in that analytical procedure is as follows: the cDNA that, utilizes detected prokaryotic organism RNA to obtain for the template reverse transcription carries out gradient dilution and draws out the doubling dilution typical curve, obtain slope slope, by PCR effectiveness formula E=10 ^[1/slope]-1 calculates Real-time PCR efficient E; Two, the extraction of procaryotic total nucleic acid: utilize phenol-chloroform method to extract and be in the logarithmic growth detected procaryotic total nucleic acid in mid-term; Three, the total nucleic acid of being extracted is prepared reverse transcription sample and non-reverse transcription sample, the preparation method is as follows: the detected prokaryotic organism total nucleic acid through the step 2 extraction of getting two equal portions; Wherein portion is carried out reverse transcription reaction obtain the reverse transcription sample; Is that 0.1% DEPC water is diluted to the concentration identical with the RT sample with another part with volumetric concentration, obtains non-reverse transcription sample; Four, reverse transcription sample and the non-reverse transcription sample that obtains in step 3 carries out Real-time PCR detection, will obtain the Ct of reverse transcription sample in the step 3 respectively _RTThe Ct of value and non-reverse transcription sample _RT-value; Five, the analysis expressed of prokaryotic gene relative quantification: specify that one group of gene expression amount Q ' passes through the relative quantification formula for the data that reference group adopts above-mentioned steps to record in the detected prokaryotic organism $Q_R=\frac{Q}{Q^{\&prime;}}=\frac{{(1+E)}^{-\mathrm{\&Delta;Ct}}-1}{{(1+E)}^{{-\mathrm{\&Delta;Ct}}^{\&prime;}}-1}$ Calculate the relative expression quantity Q of prokaryotic gene _RWherein Q represents the expression amount of goal gene, the expression amount of Q ' expression reference gene, $\mathrm{\&Delta;Ct}={\mathrm{Ct}}_{\mathrm{RT}}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}},$ ${\mathrm{\&Delta;Ct}}^{\&prime;}={\mathrm{Ct}}_{\mathrm{RT}}^{\&prime;}-{\mathrm{Ct}}_{{\mathrm{RT}}^{-}}^{\&prime;},$ E=10 ^[1/slope]-1, the Ct value is meant the cycle number when fluorescent signal reaches threshold value, and RT represents reverse transcription, RT ^-Represent non-reverse transcription.

2, a kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression according to claim 1, it is characterized in that the slope calculations slope method in the described step 1 is as follows: cDNA is carried out 5 10 times of gradient dilutions respectively, carry out Real-time PCR then and detect; If the minimum template concentrations of dilution is 10 ¹, with the logarithmic value 1g10 of template concentrations multiple ¹～1g10 ⁵Be X-coordinate, the Ct value is an ordinate zou drawing standard curve, obtains slope slope.

3, the relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression according to claim 1, it is as follows to it is characterized in that extracting the total nucleic acid method in the described step 2: the bacterial strain that activates bacterium earlier, bacterial isolates after will activating then is inoculated in the substratum, cultivate logarithmic growth during mid-term, thalline is collected in the centrifugation of bacterium liquid, utilized conventional phenol-chloroform method to extract total nucleic acid again.

4, a kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression according to claim 1, it is characterized in that the reverse transcription sample preparation methods is as follows in the described step 3: the step 2 of learning from else's experience method is extracted in the detected prokaryotic organism total nucleic acid and is carried out reverse transcription reaction, the temperature of reverse transcription is 42 ℃, the time of reverse transcription is 15min, the temperature of ThermoScript II inactivation is 95 ℃, the time of ThermoScript II inactivation is 2min, obtains the reverse transcription sample.

5, a kind of relative quantification method of utilizing the real-time fluorescent reverse transcription PCR analyzing prokaryote gene expression according to claim 1, it is characterized in that in the described step 4 detected procaryotic reverse transcription sample and non-reverse transcription sample being carried out pcr amplification, after the PCR reaction conditions is 95 ℃ of sex change 10s, 95 ℃ of 5s, 40 circulations of 60 ℃ of 34s amplifications, to when 60 ℃ of 34s, collect fluorescent signal and be set at threshold value, measure the Ct of reverse transcription sample more respectively _RTThe Ct of value and non-reverse transcription sample _RT-Value.