Method for detecting transcription level of Macaca mulatta gene ABCG2 through RT-qPCR

A technology of gene transcription and gene expression level, which is applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., to achieve the effect of high specificity, strong specificity and high sensitivity

Active Publication Date: 2018-07-27
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, there is no use of rhesus monkeys at home and abroad. ABCG2 Research on Quantitative Detection Method of Transcript Level

Method used

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  • Method for detecting transcription level of Macaca mulatta gene ABCG2 through RT-qPCR
  • Method for detecting transcription level of Macaca mulatta gene ABCG2 through RT-qPCR
  • Method for detecting transcription level of Macaca mulatta gene ABCG2 through RT-qPCR

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Embodiment

[0051] A RT-qPCR detection in macaques ABCG2 A method at the level of gene transcription, comprising the steps of:

[0052] (1) Design the following primers:

[0053] According to the macaca mulatta monkey in the NCBI gene bank ABCG2 Nucleotide sequence number: NM_001032919.1, rhesus monkey ( macaca mulatta )of GAPDH Nucleotide sequence number NM_001195426.1 designed primers, the above primers were synthesized by Shanghai Bioengineering Co., Ltd.:

[0054] Macaque ABCG2 The specific upstream and downstream primers for gene expression level are:

[0055] ABCG2 F: 5’-TTAGCTGCAAGGAAAGATCCAAG-3’

[0056] ABCG2 R: 5'-GTCATAGTTGTTGGAAGCCGAAG-3'

[0057] The specific upstream and downstream primers of the rhesus monkey GAPDH gene as an internal reference gene are:

[0058] GAPDH F: 5'-AGCCCCATCACCATCTTCC-3'

[0059] GAPDH R: 5'-AATGAGCCCCAGCCTTCTC-3';

[0060] Macaque ABCG2 The primer sequences and fragment sizes for quantification of gene expression levels are...

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Abstract

The present invention provides a method for detecting the transcription level of a Macaca mulatta gene ABCG2 through RT-qPCR. According to the method, cDNA obtained through the reverse transcription synthesis of total RNA extracted from the fresh kidney tissue of Macaca mulatta is used as a template, real-time fluorescence quantitative PCR amplification is performed with a PCT primer combination,the Ct values, the dissolving peaks and the amplification efficiencies of the gene ABCG2 fragment and the internal reference gene GAPDH fragment are obtained, and the [delta][delta]C(t) value expressed by the normalized gene ABCG2 folds is obtained through the conventional treatment so as to obtain the relative expression value of the gene ABCG2 transcription level. According to the present invention, the method provides the effective way for the research on the function of Macaca mulatta gene ABCG2 and the influence caused by related drugs, is suitable for the real-time quantitative PCR detection, and has advantages of simple operation, high reproducibility, low detection cost, high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to a detection method, in particular to a real-time fluorescent quantitative RT-qPCR method for detection of macaque adenosine triphosphate binding cassette transporter ABCG2 The method for gene transcription level belongs to the field of molecular biotechnology. Background technique [0002] In recent years, genome-wide association studies (GWAS) have detected multiple susceptibility loci and related candidate genes that lead to hyperuricemia and gout. in SLC2A9 , SLC22A11 and SLC22A12 Loss-of-function mutations in genes cause hereditary hypouricemia, while overexpression enhances uric acid reabsorption. ABCG2, SLC17A1 and SLC17A3 Defective gene function variants reduce renal and intestinal excretion of uric acid. As a transport protein, ABCG2 is located in the cell membrane and is widely distributed in tissues with secretion and excretion functions. In the kidney, ABCG2 is located in the brush border of the renal pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2600/158C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 唐东红王陈芸叶尤松李哲丽马开利鲁帅尧杨浩陈倩肖涵邱炳玲杨忠
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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