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Real-time fluorescence quantitative PCR analysis method for watermelon gene expression

A real-time fluorescence quantification and gene expression technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of wrong results and overestimation of gene expression levels, and achieve great practicability, ensure accuracy, and save money. The effect of the cost of experiments

Inactive Publication Date: 2015-06-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the influence of gDNA contamination and pseudogenes cannot be ruled out during gene expression analysis, so that the gene expression level is overestimated, resulting in erroneous results

Method used

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  • Real-time fluorescence quantitative PCR analysis method for watermelon gene expression
  • Real-time fluorescence quantitative PCR analysis method for watermelon gene expression
  • Real-time fluorescence quantitative PCR analysis method for watermelon gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 The design and expression analysis of primers at junction sites of adjacent exons in watermelon CCD1 gene

[0023] (1) Acquisition of watermelon CCD1 gene sequence and structure information:

[0024] Sequence references of carotenoid hydroperoxide lyase gene (CCD1) Grassi S, Piro G, Lee J M, et al.Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit[J].BMC genomics,2013,14( 1): 781. Download the gene sequence and gene structure information (ID: Cla015245) from Cucurbitaceae Genome Database (http: / / www.icugi.org / ), see SEQ ID NO.5 for the gene sequence, and see figure 1 .

[0025] (2) Design of primers for random binding sites of CCD1 gene:

[0026] When performing gene expression analysis, in the process of designing primers for the target gene, the CDS sequence of the gene is usually placed in the primer design software for primer design. This method does not consider the binding site of the primer on the gene. ...

Embodiment 2

[0041] Example 2 Design and expression analysis of watermelon CHYB junction site primers spanning adjacent exons

[0042] (1) Obtaining the sequence and structure information of watermelon CHYB gene:

[0043] Watermelon β-carotene hydroxylase gene (CHYB) gene sequence references Grassi S, Piro G, Lee J M, et al.Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit[J].BMC genomics,2013,14( 1): 781. The gene sequence and gene structure information (ID: Cla006149) were downloaded from Cucurbitaceae Genome Database (http: / / www.icugi.org / ). For the gene sequence, see SEQ ID NO.6, and for the structural information, see Figure 6 .

[0044] (2) Design of primers for random binding sites of CHYB gene:

[0045] The method refers to step (2) in Example 1, and the primer is named as CHYB', and its sequence is shown in Table 1. After sequence alignment, it was found that the CHYB' primer pair was located on the first exon.

[0046] (3) Pr...

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Abstract

The invention discloses a real-time fluorescence quantitative PCR analysis method for watermelon gene expression, belonging to the technical field of gene expression analysis. The real-time fluorescence quantitative PCR analysis method for watermelon gene expression comprises the following steps: according to the structure information of a target gene, specifically designing the binding site of a forward or reverse primer of the target gene on the joint of the adjacent exons so as to be amplified to a target fragment on a cDNA template and not to be amplified to a product on a genome DNA template, and then performing real-time fluorescence quantitative PCR analysis for the target gene expression through using the primer. The primer designing method can be used for excluding the influence of genome DNA pollution in a cDNA sample on the gene expression result, and the watermelon gene expression data is more accurate by utilizing the real-time fluorescent quantitative PCR technology. The real-time fluorescence quantitative PCR analysis method not only is wide in usage range, but also can be used for saving the experimental cost, and has great practicability.

Description

technical field [0001] The invention belongs to the technical field of gene expression analysis, in particular to a real-time fluorescence quantitative PCR analysis method for gene expression of watermelon. Background technique [0002] Real-time fluorescent quantitative PCR (qRT-PCR for short) is a commonly used technique in the quantitative analysis of gene expression and an indispensable and important research method in molecular biology research. It is based on the polymerase chain reaction (polymerase chain reaction). chain reaction, PCR) and reverse transcription technology developed. [0003] In the process of gene expression analysis, the RNA of the sample must first be extracted, then reverse-transcribed into cDNA, and then the cDNA is used as a template, and the gene expression is quantitatively analyzed by qRT-PCR technology using the specific primers of the target gene. Since there is usually genomic DNA contamination in the process of extracting RNA, relevant e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q1/6848C12Q2600/158C12Q2531/113
Inventor 别之龙孔秋生高凌云曹蕾袁静贤
Owner HUAZHONG AGRI UNIV