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Primers, kits and methods for RT-qPCR detection of liver purine nucleoside phosphatase PNP gene transcription level in macaques
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A technology of purine nucleoside phosphatase and gene transcription, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of strong specificity, simple method and high repeatability
Active Publication Date: 2021-04-27
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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At present, there is no research on the quantitative detection method of PNP transcription level using rhesus monkeys at home and abroad
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Embodiment 1
[0059] Primers for RT-qPCR detection of macaque hepatic purine nucleoside phosphatase PNP gene transcription level, including the specific upstream and downstream primer pairs of rhesus monkey PNP gene expression level and the specific upstream and downstream primer pairs of rhesus monkey GAPDH gene as an internal reference gene;
[0060] Among them, the specific upstream and downstream primers for the expression level of the macaque PNP gene 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 macaque liver, comprising the primers described in Example 1, SYBR Premix Ex Taq II enzyme, cDNA template of macaque 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 PNP nucleotide sequence number of macaca (macaca mμlatta) in the NCBI gene bank: NM_001193551, the GAPDH nucleotide sequence number of macaca (macaca mμlatta) NM_001195426.1 designed primers, and the above primers were synthesized by Shanghai Bioengineering Co., Ltd.:
[0072] The specific upstream and downstream primers for the expression level of macaque PNP gene 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] The primer sequences and f...
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Abstract
The invention relates to a primer, a kit and a method for detecting the transcription level of purine nucleoside phosphatase PNP gene in rhesus monkey liver by RT-qPCR, and belongs to the field of molecular biology technology. The method of the present invention uses the cDNA synthesized by reverse transcription of the total RNA extracted from the fresh liver tissue of rhesus monkey as a template, and uses the PCR primer combination to perform real-time fluorescent quantitative PCR amplification to obtain PNP Gene fragments and reference genes GAPDH The Ct value, dissolution peak, and amplification efficiency of the fragments were normalized by routine processing. PNP The ΔΔC(t) value of the gene fold expression, thus obtaining PNP Relative expression values of gene transcript levels. The invention provides an effective way for studying the PNP function of macaques and the influence of related drugs on it. At the same time, the invention is suitable for real-time quantitative PCR detection, and has simple operation, high repeatability, low detection cost, high sensitivity and strong specificity. Easy to promote applications.
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...
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