Method for detecting whether genome DNA contamination exists in cDNA sample of watermelon

Abstract

The invention belongs to the technical field of gene expression analysis and discloses a method for detecting whether genome DNA contamination exists in a cDNA sample during watermelon gene expression analysis. The principle of the method is as follows: according to structure and sequence information of genes, forward and reverse primers are designed respectively on two adjacent exons, so that amplification products on a genome DNA template are larger than those on a cDNA template. According to the principle, specific primers of molecular chaperone DnaJ protein gene DNAJ widely existing in a watermelon are designed, the cDNA sample is taken as a template, PCR (polymerase chain reaction) amplification is performed by the aid of the primer pair, if amplification products larger than 87bp appear, the phenomenon indicates that the genome DNA contamination exists in the cDNA sample, and if only amplification products of 87bp appear, the phenomenon indicates that no genome DNA contamination exists in the cDNA sample. With the adoption of the method, the accuracy of watermelon gene expression analysis with technologies of real-time fluorescent quantitative PCR and the like can be further improved.

Description

technical field [0001] The invention belongs to the technical field of gene expression analysis, and relates to a method for detecting whether there is genomic DNA pollution in a cDNA sample when analyzing watermelon gene expression. Background technique [0002] Gene expression analysis has been widely used in the fields of gene function identification and transcriptional regulation. At present, the commonly used gene expression analysis techniques include semi-quantitative RT-PCR and real-time fluorescent quantitative PCR. Among them, real-time fluorescent quantitative PCR technology has become the main gene expression analysis technology because of its advantages of fast, accurate and less required sample volume. [0003] The sample preparation stage is the same whether semi-quantitative RT-PCR or real-time PCR is used for gene expression analysis. First, sample RNA is extracted, reverse-transcribed into cDNA, and then semi-quantitative RT-PCR or real-time fluorescent qu...

AI Technical Summary

Problems solved by technology

But there is no way to detect whether the genomic DNA in the cDNA sample is completely removed

Moreover, in actual operation, the step of degrading genomic DNA may be missed, or due to the influence of factors such as enzyme activity and reaction conditions, genomic DNA may not be removed, which will seriously affect the accuracy of subsequent gene expression analysis.

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