Internal reference gene and construction method for PCR expression analysis of Caucasian clover

Abstract

The invention discloses an internal reference gene and a construction method for PCR (polymerase chain reaction) expression analysis of caucasian clover. A nucleotide sequence of the internal reference gene is as shown in the sequence SEQ ID No.2; the construction method comprises the steps as follows: selecting 7 internal reference genes with stable expression by utilizing 7 tissue transcriptomedata of caucasian clover, and selecting one traditional internal reference gene commonly used in clover as a control; performing primer design on the candidate internal reference genes, performing PCRamplification on each cDNA, and obtaining the amplification efficiency of primers and the cycle number of amplification reaching a threshold value; evaluating and analyzing the obtained data for internal reference stability, and finally screening the most suitable internal reference gene and the number of the internal reference gene under specific experimental conditions. The reference gene is used as a reference for detecting the gene expression level change of caucasian clover, and for correcting the loading quantity of sample and an experimental error existing in a sample loading process,so as to ensure the accuracy of experimental results.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an internal reference gene and a construction method for PCR expression analysis of Caucasus clover. Background technique [0002] Gene expression analysis is widely used in the field of life sciences, and its in-depth research is crucial to exploring disease-related genes and regulatory mechanisms and revealing the mysteries of life. Quantification of gene expression at the transcriptional level, such as real-time fluorescent quantitative PCR (Quantitative real-time RT-PCR, qRT-PCR), Northern blotting (Northern blotting), ribonuclease protection assay (ribonuclease protection assay, RPA), gene chip (gene chip), etc., in order to obtain more accurate and reliable results, it is necessary to standardize the expression level of the target gene with the internal reference gene. [0003] Polymerase chain reaction technology (polymerase chain reaction, referred to as PCR tech...

AI Technical Summary

Problems solved by technology

At present, traditional internal reference genes are mostly used in gene expression analysis of Clover plants. These traditional internal reference genes have not been systematically verified and have poor stability, resulting in poor accuracy of gene expression analysis of Clover plants.

In reality, there is no ideal internal reference gene with constant expression under different experimental conditions

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