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Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof

A technology of internal reference gene and gene chip, which is applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc.

Active Publication Date: 2014-03-19
GUIZHOU TOBACCO SCI RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional internal reference genes are determined through literature search and prediction, and then verify the relative stability of the genes through experiments, which has great limitations

Method used

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  • Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof
  • Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof
  • Tobacco reference genes screened by use of gene chip and combination of real time PCR (polymerase chain reaction) and method thereof

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Experimental program
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Embodiment Construction

[0028] The present invention will be described in detail below in conjunction with specific embodiments.

[0029] 1 test sampling

[0030] 1.1 Experimental material: Tobacco cultivar K326

[0031] 1.2 Experimental materials: roots, stems, and leaves were taken from tobacco leaves when transplanting, 20 days, 40 days, 60 days, 70 days (10 days after topping), and 100 days (40 days after topping) respectively. , stems and roots. Tobacco flowers were sampled at budding, 5 days after budding, and 10 days after budding, and all harvested tissues were immediately frozen in liquid nitrogen and stored at -80°C until use.

[0032] 2 chip hybridization

[0033] 2.1 The main technical process of the experiment is as follows:

[0034] (1) Extract the total RNA of tobacco samples by Trizol method;

[0035] (2) Reverse transcription to synthesize single-stranded cDNA;

[0036] (3) Fluorescence labeling and purification of labeled cDNA;

[0037] (4) chip hybridization;

[0038] (5) c...

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Abstract

The invention discloses tobacco reference genes screened by use of a gene chip and combination of a real time PCR (polymerase chain reaction) and a screening method thereof. By use of the screening method, the new tobacco reference genes comprising tobacco ribulose phosphate kinase gene and tobacco digalactosyl diacylglycerol synthase gene, the stability of the tobacco ribulose phosphate kinase gene is higher than that of all reported tobacco reference genes in the prior art, and the stability of the tobacco digalactosyl diacylglycerol synthase gene is next only to that of reported tobacco reference gene EF-1-a.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for screening tobacco internal references by using a gene chip combined with Real time PCR. Background technique [0002] Using real time PCR to analyze gene expression levels is an important research method in molecular biology, and the key to the implementation of this technology is to select appropriate internal references, so the selection of internal references has always been a hot spot for researchers. The traditional determination of internal reference genes is done through literature search and prediction, and then through experiments to verify the relative stability of genes, which has great limitations. [0003] Therefore, there are defects in the prior art and need to be improved. Contents of the invention [0004] The technical problem to be solved by the present invention is to provide a method and application for screening tobacco internal r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2531/113C12Q2561/113
Inventor 郭玉双任学良黎瑞源
Owner GUIZHOU TOBACCO SCI RES INST
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