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Method for detecting transcription level of tree shrews gene SLC22A12/UTAT1 through RT-qPCR

A technology of gene transcription and tree shrew, applied in the field of detection, to achieve the effect of simple method, strong specificity and high sensitivity

Pending Publication Date: 2018-07-27
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

At present, there is no research on the quantitative detection method of SLC22A12 / URAT1 transcription level using tree shrews at home and abroad

Method used

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  • Method for detecting transcription level of tree shrews gene SLC22A12/UTAT1 through RT-qPCR
  • Method for detecting transcription level of tree shrews gene SLC22A12/UTAT1 through RT-qPCR
  • Method for detecting transcription level of tree shrews gene SLC22A12/UTAT1 through RT-qPCR

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Embodiment

[0053] The method for detecting tree shrew SLC22A12 / URAT1 gene transcription level by RT-qPCR is characterized in that it comprises the following steps:

[0054] (1) Design the following primers:

[0055] The specific upstream and downstream primers for the expression level of tree shrew SLC22A12 / URAT1 are:

[0056] SLC22A12 / URAT1 F: 5'-CTACGACCACGGCACTTTCA-3'

[0057] SLC22A12 / URAT1 R: 5'-AAGGTAACTCCAGGTCAGCAC-3'

[0058] The specific upstream and downstream primers of the tree shrew GAPDH gene as an internal reference gene are:

[0059] GAPDH F: 5'-AGCCCCATCACCATCTTCC-3'

[0060] GAPDH R: 5'-AATGAGCCCCAGCCTTCTC-3';

[0061] tree shrew URAT1 (SLC22A12) The primer sequences and fragment sizes for quantification of gene expression levels are shown in Table 1:

[0062] Table 1

[0063]

[0064] (2) Using total RNA extracted from fresh tree shrew kidney tissue as a template, the first strand of cDNA from tree shrew kidney tissue was synthesized by conventional revers...

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Abstract

The present invention provides a method for detecting the transcription level of a tree shrews gene SLC22A12 / UTAT1 through RT-qPCR. According to the method, the total RNA is extracted from the fresh kidney tissue of tree shrews, conventional reverse transcription synthesis is performed to obtain a tree shrews kidney tissue cDNA first strand 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 SLC22A12 / UTAT1 fragment and the internal reference gene GAPDHfragment are obtained, and the [delta][delta]C(t) value expressed by the normalized gene SLC22A12 / UTAT1 folds is obtained through treatment so as to obtain the relative expression value of the gene SLC22A12 / UTAT1 transcription level. According to the present invention, the method provides the effective way for the research on the function of tree shrews urate anion transporter 1 and the influencecaused 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 method for detecting the transcription level of tree shrew urate anion transporter 1 (SLC22A12 / URAT1) by a real-time fluorescence quantitative RT-qPCR method, and belongs to the field of molecular biology technology. Background technique [0002] Urate anion transporter 1 (urate anion transporter 1, URAT1) is the main transporter for human renal tubular reabsorption of uric acid, and is the first protein found to be related to renal urate transport, mainly present in renal tubular epithelium On the brush border membrane of cells, it belongs to the organic anion transporter (OAT) family, and the encoding gene is SLC22A12 , by mediating the transport of uric acid from the lumen to the renal tubular epithelial cells by reabsorption (concentration gradient and chemical gradient on both sides of the lumen), and has a strong uric acid reabsorption capacity. In the body, uric acid is excreted ou...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107C12Q2545/101
Inventor 唐东红王陈芸叶尤松李哲丽马开利鲁帅尧肖涵邱炳玲陈倩杨浩杨忠
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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