Method, primers and kit for detecting PD-L1 gene expression quantity through real-time fluorescent quantitative PCR

Abstract

The invention discloses a method, primers and a kit for detecting PD-L1 gene expression quantity through real-time fluorescent quantitative PCR. The method comprises the following steps: (1) synchronously extracting DNA and mRNA in a sample; (2) synthesizing a first chain cDNA by taking the to-be-detected mRNA as a template through a special reverse transcription primer; and (3) carrying out dual-channel real-time fluorescent quantitative PCR amplification by respectively adopting a primer corresponding to the specific sequence on the reverse transcription primer and a specific primer corresponding to a reference gene and taking a reverse transcription product cDNA and reference gene DNA as templates while amplifying a target gene cDNA and a reference gene, and calculating the copy numberratio of the target gene to the reference gene in the sample according to the respective standard curves by taking a plasmid constructed by linking the reference gene and the target gene sequence as astandard substance to obtain the expression level of the target gene, so that various experimental operation errors are synchronously corrected, and the reliability and the repeatability of the result are ensured.

Description

technical field [0001] The invention relates to a method for detecting the expression level of PD-L1 gene. Background technique [0002] Programmed cell death ligand 1 (processed death-1ligand 1, PD-L1), also known as B7-H1, CD274, is an inhibitory co-stimulatory molecule encoded by the CD274 gene. PD-L1 is a type I transmembrane glycoprotein with 290 amino acids, including an extracellular region, a hydrophobic transmembrane region and a tail cytoplasmic region. PD-L1 protein is widely expressed in lymphocytes, such as activated T and B cells, macrophages, dendritic cells, thymic endothelial cells and other cells, as well as in the heart and placenta, as well as in various tumor cells and tumor microenvironments. Highly expressed in immune cells in lung cancer, esophageal cancer, breast cancer, lymphoma, etc., the PD-L1 described in the literature: Dong, H. et al. (2002) NatMed 8, 793-800, hompson, R.H. et al. ( 2006) Cancer Res 66, 3381-5. and Pardoll, D.M. (2012) Nat Re...

AI Technical Summary

Problems solved by technology

[0007] Traditional RNA expression detection methods usually use GAPDH, beta-actin, tubulin and other mRNAs as references. However, due to the different expression levels of reference genes in different tissue parts and the unstable expression caused by different growth states of cells, it is also verified by experiments. , this standard cannot completely and accurately reflect the expression level of the gene to be tested.

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