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Kit for one-step real-time fluorescent quantitative reverse transcription polymerase chain reaction detection of human miR-1290

A technology of reverse transcription polymerase and real-time fluorescence quantification, which is applied in the biological field, can solve the problem of small number of cells, achieve the effects of reducing false positive interference, avoiding cross-contamination, and improving specificity

Inactive Publication Date: 2019-03-29
安徽普元生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Surviving tumor cells in the blood circulation are the basis of distant metastasis and recurrence. Although various detection methods for various colorectal cancers continue to increase, the number of such cells is extremely small, and it is not easy to be detected by clinical routine methods

Method used

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  • Kit for one-step real-time fluorescent quantitative reverse transcription polymerase chain reaction detection of human miR-1290
  • Kit for one-step real-time fluorescent quantitative reverse transcription polymerase chain reaction detection of human miR-1290

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Effect test

Embodiment 1

[0022] One-step fluorescence quantitative RT-PCR method was used to detect the expression of miR-1290.

[0023] 1. Materials:

[0024] Trizol reagents and restriction endonucleases were purchased from Invitrogen, USA, pGEM-T-Easy cloning vector, M-MLV reverse transcriptase, Taq DNA polymerase, Oligo(dT) 15-18 Purchased from Promega Company of the United States, ABI7500 quantitative PCR instrument is a product of ABI Company of the United States.

[0025] 2. Primer and probe design and synthesis:

[0026] Using the full-length sequence of miR-1290 (GenBank accession number NC_000001.11) as a template, use PrimerExpress TM (V3.0, American ABI Company) software analyzes TaqMan primers and probe sites, and selects the best combination based on consideration of the miR-1290 genomic DNA sequence. The primers of the standard product of the present invention and the primers for inspection are the same primers.

[0027] The upstream primer sequence is

[0028] 5'-GAGCGTCACGTTGACAC...

Embodiment 2

[0037] Application of fluorescent quantitative RT-PCR method to detect miR-1290.

[0038]Peripheral blood samples from 8 patients with malignant tumors confirmed by pathology were separated. Peripheral blood nucleated cells were collected by centrifugation after washing with PBS. Total RNA was extracted with Trizol reagent, and 2.5 μl RNA was taken in a volume of 50 μl reverse transcription polymerase chain reaction. , use the upstream and downstream primers to carry out PCR amplification on the ABI 7500 PCR instrument, the conditions are reverse transcription at 45°C for 30 minutes, pre-denaturation at 95°C for 5 minutes, 95°C for 30 seconds, 60°C for 20 seconds, and 72°C for 30 seconds, a total of 40 Cycle, 72°C extension for 10 minutes followed by 4°C. Simultaneously, standard samples were added to detect the standard curve. The measurement results were processed by the instrument and calculated according to the standard curve to calculate the miR-1290 expression level of ...

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Abstract

The invention provides a kit for real-time fluorescent quantitative RT-PCR detection of human miR-1290. The kit takes total mRNA as a template, so that reverse transcription and real-time fluorescentquantitative PCR can be carried out in a same reaction system, opening of a tube cap is not needed in the reaction process, cross-contamination is avoided, and the expression quantity of human miR-1290 in a sample can be accurately quantitatively detected. The kit can be used for detecting the expression of miR-1290 in peripheral blood samples of patients in clinical and scientific research, and can be used for assistant diagnosis, guide treatment, recurrence and prognosis reminding of colorectal cancer.

Description

technical field [0001] The invention belongs to the field of biological technology, and is a reagent that can accurately and quantitatively detect the expression level of human miR-1290 in a sample by using mRNA as a template to perform reverse transcription and real-time fluorescent quantitative PCR in the same reaction system. box. Background technique [0002] In the middle of the last century, with the continuous change and improvement of people's living environment, eating habits and living standards, the morbidity / death rate of various tumors also showed an upward trend year by year. The morbidity and mortality of colorectal cancer are second only to lung and breast cancer, and it has become the third malignant tumor. Colorectal cancer has highly malignant and aggressive biological characteristics, and often infiltrates and metastasizes. This biological characteristic is an important factor leading to poor prognosis and high mortality in patients. The occurrence and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/6886
CPCC12Q1/6886C12Q1/686C12Q2600/158C12Q2600/178C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 程媛
Owner 安徽普元生物科技股份有限公司
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