Selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke

An internal reference gene, real-time quantitative technology, applied in the field of quantitative PCR

Inactive Publication Date: 2018-09-28
青海大学农林科学院
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Problems solved by technology

However, so far, no internal reference gene has been found that can fully meet the above conditions. Therefore, under specific and reproducible experimental conditions, selecting an internal reference gene with stable expression is an important prerequisite for qPCR analysis.

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  • Selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke
  • Selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke
  • Selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke

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specific Embodiment approach

[0016] combine figure 1 , figure 2 and image 3 Show the specific embodiment of the selection method of internal reference gene in a kind of Jerusalem artichoke real-time quantitative PCR analysis of the present invention: The steps of the selection method of internal reference gene in this Jerusalem artichoke real-time quantitative PCR analysis are as follows:

[0017] (1) Extraction of total RNA: Weigh 100mg of Jerusalem artichoke leaf or tuber tissue into a pre-cooled mortar, quickly grind it into powder in liquid nitrogen, and transfer it to a 1.5mL enzyme-free centrifuge tube filled with 1mL Trizol , place the homogenized sample at room temperature for 5min, add 200ul chloroform, cover the tube cap, shake vigorously on a vortex oscillator for about 15sec, then let it stand at room temperature for 3min, 12000r·min -1 Centrifuge at 4°C for 10 minutes, the sample will be divided into three layers, transfer the upper transparent aqueous phase (500ul) to a new enzyme-free c...

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Abstract

The invention discloses a selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke, and relates to the field of quantitative PCR. The method comprises the following steps: taking different tissues (radicles, immature stems, leaves, stem blocks and petals) of the jerusalem artichoke as materials, and carrying out expression analysis of the reference genes of18S ribosomal RNA gene (18S rRNA), transcription elongation factor gene (Ef-1a), actin gene (Actin and beta-actin), 3-glyceraldehyde phosphate dehydrogenase (GAPDH), 25S ribosomal RNA gene (25S rRNA)and poly-ubiquitin enzyme gene (UBQ)7 by using a q PCR technology; carrying out statistical assessment on the obtained data and analyzing expression change of all housekeeping genes by utilizing GeNorm and NormFinder software, so as to screen out relatively-stable genes as the reference genes of the jerusalem artichoke, which are used for studying the gene dosage changes of the jerusalem artichoke.

Description

technical field [0001] The invention relates to the field of quantitative PCR, in particular to a method for selecting internal reference genes in real-time quantitative PCR analysis of Jerusalem artichoke. Background technique [0002] Real-time quantitative PCR (quantitative real-time PCR, qPCR) uses a real-time fluorescence quantitative instrument to detect the amount of products after the PCR reaction, so as to achieve the purpose of nucleic acid quantification. It is more sensitive for detecting low copy number mRNA than traditional RNA quantification techniques. Realized the leap of polymerase chain reaction (polymerase chain reaction, PCR) from qualitative to quantitative, and has the advantages of high quantitative accuracy, good repeatability, strong sensitivity and fast speed. With the development of genomics and high-throughput sequencing technology, real-time fluorescent quantitative PCR has become an important tool for analyzing gene expression characteristics ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6895
CPCC12Q1/6851C12Q1/6895C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 钟启文李莉刘明池宋向阳赵孟良李屹谭龙马元鑫
Owner 青海大学农林科学院
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