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Primers, kit and method to detect transcriptional level of purine nucleotide phosphorylase PNP gene in macaque liver by RT-qPCR (real-time quantitative polymerase chain reaction)

A purine nucleoside phosphatase and gene transcription technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve high sensitivity, high repeatability, and strong specificity

Active Publication Date: 2018-11-02
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Primers, kit and method to detect transcriptional level of purine nucleotide phosphorylase PNP gene in macaque liver by RT-qPCR (real-time quantitative polymerase chain reaction)
  • Primers, kit and method to detect transcriptional level of purine nucleotide phosphorylase PNP gene in macaque liver by RT-qPCR (real-time quantitative polymerase chain reaction)
  • Primers, kit and method to detect transcriptional level of purine nucleotide phosphorylase PNP gene in macaque liver by RT-qPCR (real-time quantitative polymerase chain reaction)

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Embodiment 1

[0059] Primers for RT-qPCR detection of liver purine nucleoside phosphatase PNP gene transcription levels in macaques, including macaques PNP Specific upstream and downstream primer pairs for gene expression levels and specific upstream and downstream primer pairs for macaque GAPDH gene as an internal reference gene;

[0060] Among them, macaques PNP The specific upstream and downstream primers for gene expression level are:

[0061] PNP F: 5'-gagaccatggagaacggatacac-3'

[0062] PNP R: 5'-cagacctcctaatccagaaccac-3'

[0063] The specific upstream and downstream primers of the rhesus monkey GAPDH gene as an internal reference gene are:

[0064] GAPDH F: 5'-agccccatcaccatcttcc-3'

[0065] GAPDH R: 5'-aatgagccccagccttctc-3'.

Embodiment 2

[0067] A kit for detecting the transcription level of purine nucleoside phosphatase PNP gene in rhesus monkey liver, comprising the primers described in Example 1, SYBR Premix Ex Taq II enzyme, cDNA template of rhesus monkey liver tissue and deionized water.

Embodiment 3

[0069] A method for RT-qPCR detection of macaque liver purine nucleoside phosphatase PNP gene transcription level, comprising the following steps:

[0070] (1) Design the following primers:

[0071] According to the macaque in the NCBI gene bank ( macaca mulatta )of PNP Nucleotide sequence number: NM_001193551, rhesus monkey ( macaca mulatta )of GAPDH Nucleotide sequence number NM_001195426.1 designed primers, the above primers were synthesized by Shanghai Bioengineering Co., Ltd.:

[0072] Macaque PNP The specific upstream and downstream primers for gene expression level are:

[0073] PNP F: 5'- gagaccatggagaacggatacac-3'; (SEQ ID NO.1)

[0074] PNP R: 5'-cagacctcctaatccagaaccac-3'; (SEQ ID NO.2)

[0075] The specific upstream and downstream primers of the rhesus monkey GAPDH gene as an internal reference gene are:

[0076] GAPDH F: 5'-agccccatcaccatcttcc-3'; (SEQ ID NO.3)

[0077] GAPDH R: 5'-aatgagccccagccttctc-3'. (SEQ ID NO.4)

[0078] Macaque ...

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Abstract

The invention relates to primers, kit and method to detect transcriptional level of purine nucleotide phosphorylase PNP gene in macaque liver by RT-qPCR (real-time quantitative polymerase chain reaction), and belongs to the technical field of molecular biology. A template is made with cDNA synthesized by reverse transcription of total RNA extracted from fresh macaque liver tissue; a PCR primer combination is used to perform real-time fluorescent quantitative PCR amplification; Ct values of PNP gene fragments and reference gene GAPDH fragments, dissolution peaks, and amplification efficiency are attained; multiple-expressed delta delta C(t) value of the homogenize PNP gene is acquired by conventional treatment; relative expression value of the transcriptional level of PNP gene is acquired finally. An effective means to study PNP functionality of macaque and influence of relevant drugs upon PNP is provided; in addition, the primers, kit and method herein are applicable to real-time quantitative PCR detection and have the advantages of good operational simplicity, high repeatability, low detection cost, high sensitivity, high specificity and good convenience of popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method, in particular to a method for detecting the transcription level of purine nucleoside phosphatase PNP gene in rhesus monkey liver by real-time fluorescence quantitative RT-qPCR method. Background technique [0002] Purine nucleoside phosphorylase (PNP or PNPase for short) is an enzyme involved in purine metabolism. The enzyme catalyzes the conversion of inosine, xanthine, and guanosine into hypoxanthine, xanthine, and guanine. Inosine is the starting material for the synthesis of uric acid in the body. It is hydrolyzed by PNP enzyme to form hypoxanthine, which is then oxidized by xanthine oxidase (XO) to form xanthine and then uric acid. [0003] In recent years, with the improvement of people's living standards, dietary structure and living habits have changed, and the prevalence of hyperuricemia (HUA) has increased year by year. Epidemiological resea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6883C12N15/11
CPCC12Q1/6851C12Q1/6883C12Q2531/113C12Q2521/107C12Q2561/113
Inventor 唐东红王陈芸叶尤松李哲丽马开利鲁帅尧易红昆黄璋琼
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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