Method for detecting transcription level of SLC2A9/GLUT9 gene of tree shrews through RT-qPCR

A technology of gene transcription and gene expression level, applied in the field of detection, to achieve the effect of simple method, high sensitivity and high repeatability

Pending Publication Date: 2019-03-01
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

At present, there is no use of tree shrews at home and abroad. SLC2A9 / GLUT9 Research on Quantitative Detection Method of Transcript Level

Method used

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  • Method for detecting transcription level of SLC2A9/GLUT9 gene of tree shrews through RT-qPCR
  • Method for detecting transcription level of SLC2A9/GLUT9 gene of tree shrews through RT-qPCR
  • Method for detecting transcription level of SLC2A9/GLUT9 gene of tree shrews through RT-qPCR

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Embodiment

[0051] A RT-qPCR detection tree shrew SLC2A9 / The method of GLUT9 gene transcription level, comprises the following steps:

[0052] (1) Design the following primers:

[0053] According to the common sequence numbers of human, mouse and macaque SLC2A9 / GLUT9: human NM_020041.2, NM_001001290.1; mouse NM_001102414.1, NM_001012363.2, NM_001102415.1, NM_145559.2; macaque XM_015137992.1 primers were designed. of GAPDH Nucleotide sequence design primers were used as internal reference genes, gene accession number NM_001195426.1, primers were designed with Primer premier 5.0 software, and synthesized by Shanghai Bioengineering Co., Ltd.:

[0054] tree shrew SLC2A9 / GLUT 9 The specific upstream and downstream primers for gene expression level are:

[0055] SLC2A9 / GLUT9 F: 5'-ACAATGAAGCAGGAGCGACA-3'

[0056] SLC2A9 / GLUT9 R: 5'-ACTCAGGGTGATGTACGGGA-3'

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

[0058...

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Abstract

The invention provides a method for detecting the transcription level of an SLC2A9 / GLUT9 gene of tree shrews through RT-qPCR. Total RNA extracted from fresh kidney tissue of tree shrews is used as a template, and a first chain of cDNA of kidney tissue of the tree shrews is synthesized through conventional reverse transcription; qPCR amplification is performed by use of PCR primer combination, Ct values, dissolution peaks and amplification efficiency of SLC2A9 / GLUT9 gene segments and reference gene GAPDH segments are obtained, homogenized deltadelta C(t) values of SLC2A9 / GLUT9 gene multiple expression are obtained through treatment, so that the relative expression value of the transcription level of the SLC2A9 / GLUT9 gene is obtained. An effective way is provided for studying functions of GLUT9 and effects of related drugs on GLUT9. The method is applicable to real-time quantitative PCR detection and is high in sensitivity, high in specificity, simple to operate and high in reliability.

Description

technical field [0001] The invention relates to a detection method, in particular to a real-time fluorescence quantitative qPCR method for detecting tree shrew glucose transporter 9 ( SLC2A9 / A method for gene transcription level of GLUT9) 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. GLUT9 is a fructose transporter that belongs to SLC2A9 The family, located on the basolateral membrane of renal tubular epithelial cells, is mainly responsible for the secretion of uric acid to the outside of the cell. It is...

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6888
CPCC12Q1/6851C12Q1/6888C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 唐东红叶尤松王陈芸李哲丽肖涵邱炳玲陈倩杨浩杨忠马开利鲁帅尧
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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