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Ribonucleotide reductase subunit M1 gene expression fluorescence quantitative PCR detection kit

A fluorescence quantitative and gene detection technology, applied in the field of molecular biology, can solve the problems of drug loss of activity, gemcitabine resistance, expression increase, etc., and achieve the effect of improving detection sensitivity and specificity, high sensitivity and low false positive

Active Publication Date: 2014-08-06
广州达健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One possible reason is that gemcitabine can irreversibly combine with RRMI to inactivate the enzyme. In order to maintain the activity and amount of the required RR holoenzyme, the negative feedback regulation mechanism in the cell promotes the expression of RRM1, and the drug itself also loses its activity. , making tumor cells resistant to gemcitabine

Method used

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  • Ribonucleotide reductase subunit M1 gene expression fluorescence quantitative PCR detection kit
  • Ribonucleotide reductase subunit M1 gene expression fluorescence quantitative PCR detection kit
  • Ribonucleotide reductase subunit M1 gene expression fluorescence quantitative PCR detection kit

Examples

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Effect test

Embodiment 1

[0042] Example 1: The RRM1 gene expression fluorescent quantitative PCR kit of the present invention detects two genotypes of A / C and A / A at the -37 site of the RRM1 gene in tissues of patients with non-small cell lung cancer.

[0043] One probe and one pair of common primers can be designed to specifically detect the A / C and A / A genes at the -37 site.

[0044] The forward primer sequence is:

[0045] SEQ ID NO: 1 (5'-AAGAG CAGCG TGCCA GAGAT-3');

[0046] The reverse primer sequence is:

[0047] SEQ ID NO:2 (5'-ACACA TCAAA GACCA GTCCT GATTA G-3');

[0048] The fluorescent probe sequence is:

[0049] SEQ ID NO: 3 (FAM-TTGC TCTTT GGATT CCGGA TCTCT TCA-BHQ1). The 5' end of the fluorescent probe is labeled with FAM (fluorescence reporter group), and the 3' end is labeled with BHQ1 (fluorescent quencher group).

[0050] Then optimize the reaction system for FQ-PCR detection: the reaction system is 15 μl, forward and reverse primers 0.15 μl (20 μΜ), each probe 0.2 μl (20 μΜ), t...

Embodiment 2

[0053] Example 2: The RRM1 gene expression fluorescent quantitative PCR kit of the present invention detects two genotypes of C / T and C / A at the -524 site of the RRM1 gene in tissues of patients with non-small cell lung cancer.

[0054] One probe and one pair of common primers can be designed to specifically detect the -524 site to detect C / T and C / A genes.

[0055] The forward primer sequence is:

[0056] SEQ ID NO: 1 (5'-AAGAG CAGCG TGCCA GAGAT-3');

[0057] The reverse primer sequence is:

[0058] SEQ ID NO:2 (5'-ACACA TCAAA GACCA GTCCT GATTA G-3');

[0059] The fluorescent probe sequence is:

[0060] SEQ ID NO: 3 (FAM-TTGC TCTTT GGATT CCGGA TCTCT TCA-BHQ1). The 5' end of the fluorescent probe is labeled with FAM (fluorescence reporter group), and the 3' end is labeled with BHQ1 (fluorescent quencher group).

[0061] Then optimize the reaction system for FQ-PCR detection: the reaction system is 15 μl, forward and reverse primers 0.15 μl (20 μΜ), each probe 0.2 μl (20 μ...

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Abstract

The invention relates to a fluorescence quantitative PCR detection kit used for detecting ribonucleotide reductase subunit M1 gene expression. The ribonucleotide reductase subunit M1 gene expression fluorescence quantitative PCR detection kit provided by the invention comprises a fluorescence quantitative reaction solution premix, a fluorescence quantitative reaction solution, and a positive control. The fluorescence quantitative reaction solution comprises a pair of PCR primer and a probe. The fluorescent probe is a fluorescently labeled locked-nucleotide probe covering a ribonucleotide reductase subunit M1 gene detection site. A 5' terminal is labeled by a fluorescent reporter group, and a 3' terminal is labeled by a fluorescent quenching group. The positive control is a hybrid plasmid genome DNA comprising the detected ribonucleotide reductase subunit M1 gene detection site. The kit is suitable to be used in RRM1 expression level mRNA quantitative detections of various tissues, cells, and bloods.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a fluorescent quantitative PCR kit for detecting nucleotide reductase subunit M1 (RRM1) gene expression, which is suitable for quantitative detection of RRM1 expression level mRNA in various tissues, cells and blood. Background technique [0002] Ribonuleotide reductase subunit M1 (ribonuleotide reductase subunit M1, RRM1) is one of the targets of gemcitabine. Gemcitabine is an analogue of deoxycytidine nucleoside, which is a cell cycle-specific antineoplastic drug and mainly acts on DNA synthesis. Cells in the S phase can also prevent the progression from the G phase to the S phase under certain conditions. Gemcitabine is a nucleotide reductase inhibitor, which reduces the production of dCTP required for DNA synthesis in cells and inhibits DNA synthesis. Studies have confirmed that high-level expression of RRM1 is associated with gemcitabine resistance. Moreover, patients with a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 邵琦
Owner 广州达健生物科技有限公司
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