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

A prokaryotic, relatively quantitative technology, applied in the fields of basic biology and medicine, can solve the problems of large differences in PCR efficiency, unstable expression of housekeeping genes, large error in results, etc., achieve objective and accurate detection results, shorten detection time and cost, the effect of reducing inspection costs

Active Publication Date: 2008-04-30
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0007] The invention provides a relative quantitative method for analyzing prokaryotic gene expression by real-time fluorescence reverse transcription PCR, the purpose is to solve DNA residues, housekeeping gene expression instability in the growth process of bacteria and when PCR efficiency is not high or multiple When there is a large difference in PCR efficiency between genes, the error of the analyzed results is large, which leads to the problem that the relative quantification of gene expression has certain limitations in practical applications.

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  • Relative quantitative method for analyzing prokaryote gene expression by utilizing real time fluorescence reverse transcription PCR
  • Relative quantitative method for analyzing prokaryote gene expression by utilizing real time fluorescence reverse transcription PCR
  • Relative quantitative method for analyzing prokaryote gene expression by utilizing real time fluorescence reverse transcription PCR

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specific Embodiment approach 1

[0017] Specific embodiment one: In this embodiment, the relative quantitative method of prokaryotic gene expression is analyzed by real-time fluorescent reverse transcription PCR. This method can carry out relative quantitative analysis of different genes at one time. The analysis method is through the following steps Completed: 1. Use the detected prokaryotic RNA as the template to reverse transcribe the cDNA obtained by serial dilution to draw a doubling dilution standard curve, obtain the slope slope, and use the PCR efficiency formula E=10 [-1 / slope] -1 calculates Real-time PCR efficiency E; Two, the extraction of the total nucleic acid of prokaryote: utilize phenol-chloroform method to extract the total nucleic acid (DNA and RNA) of the detected prokaryote that is in logarithmic growth phase; Three, to all The extracted total nucleic acid is used to prepare a reverse transcription sample and a non-reverse transcription sample. The preparation method is as follows: take two...

specific Embodiment approach 2

[0018] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the calculation slope method in the step one is as follows: the cDNA is respectively subjected to 5 10-fold gradient dilutions, and then Real-time PCR detection is performed; The minimum template concentration is 10 1 , as the logarithmic value of the multiple of the template concentration (lg10 1 ~lg10 5 ) is the abscissa, and the Ct value is the ordinate to draw a standard curve to obtain the slope slope. Other reaction steps are the same as in Embodiment 1.

specific Embodiment approach 4

[0019] Embodiment 4: The difference between this embodiment and Embodiment 1 is that the method for extracting total nucleic acid in the step 2 is as follows: first activate the bacterial strain of the gene, and then inoculate the activated bacterial strain in the culture medium, and in a suitable bacterial When the strain was cultured to the mid-logarithmic growth phase at the growth temperature of the strain, the bacterial liquid was centrifuged to collect the bacterial cells, and then the total nucleic acid was extracted by the conventional phenol-chloroform method. Other reaction steps are the same as in Embodiment 1.

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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

technical field [0001] The invention belongs to the fields of basic biology and medicine, and relates to a relative quantitative analysis method for prokaryotic gene expression, in particular to a relative quantitative method for analyzing prokaryotic gene expression by real-time fluorescence reverse transcription PCR. Background technique [0002] With the continuous development of biological genomics and bioinformatics and the deepening of functional genomics research, gene expression research has become the research focus in recent years. Prokaryotes are an important part of the entire biological world and are closely related to human survival activities. The corresponding research has gradually shifted from structural genomics to exploratory research in the field of functional genomics, thereby finally realizing the establishment of prokaryotic systems biology. The current research on gene expression in prokaryotes is mainly analyzed from the level of gene transcription,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 姜毓君相丽闫冰毕宇涵曲妍妍赵凤刘伟
Owner NORTHEAST AGRICULTURAL UNIVERSITY