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Special primer, kit and method for testing minRNA-128 in colorectal cancer serum

A technology of mir-128 and colorectal cancer, which is applied in the field of detection of serum miRNA-128 expression in colorectal cancer, can solve the problems of miRNAs molecular loss, experimental error, and expensive reagents, and achieve simplified operation steps, reliable result analysis, and reduced The effect of testing costs

Active Publication Date: 2013-05-01
SHANDONG UNIV QILU HOSPITAL
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  • Abstract
  • Description
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Problems solved by technology

Usually, it is necessary to separate and extract the miRNAs in the serum samples by using this method, and then carry out reverse transcription and PCR amplification detection on the isolated miRNAs. Inevitably causing the loss of miRNAs molecules, causing errors in the experiment

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  • Special primer, kit and method for testing minRNA-128 in colorectal cancer serum
  • Special primer, kit and method for testing minRNA-128 in colorectal cancer serum
  • Special primer, kit and method for testing minRNA-128 in colorectal cancer serum

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Embodiment 1

[0041] (1) Composition and preparation of the kit:

[0042] The sequences of the primers are based on the nucleotide sequences reported in the microRNA database (http: / / www.mirbase.org / ) miR-128 (MIMAT0000424), miRNA-16 (MIMAT0000069) and the U6 sequence (X07425. 1) Self-designed templates, whose primer sequences and Tm values ​​are shown in Table 1.

[0043] Table 1 Primer sequence, Tm value

[0044]

[0045] The special kit also includes an RNA isolation solution, which consists of 2.5% Tween 20, 50mmol / L tris, 1mmol / L ethylenediaminetetraacetic acid and 1% bovine serum albumin.

[0046] The kit also includes a PCR reaction liquid: composed of 1×Syber Green I fluorescent dye, DNA polymerase, dNTPs, trishydrochloride, potassium chloride and magnesium chloride.

[0047] The final concentration of the DNA polymerase in the PCR reaction solution is 100U / ml.

[0048] The final concentration of the dNTPs in the PCR reaction solution is 0.2mM.

[0049] The final concentratio...

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Abstract

The invention discloses a special primer for testing the minRNA-128 in colorectal cancer serum. The special primer comprises a reverse transcription primer and a test primer of the miR-128, a reverse transcription primer and a test primer of U6 and a reverse transcription primer and a test primer of miRNA-16, as shown by SEQ IDNO: 1-9. A special kit for testing colorectal cancer serum minRNA-128 comprises the primers and a RNA separation solution. A method for testing the expression amount of the colorectal cancer serum minRNA-128 comprises steps: mixing a serum specimen to be tested with an isovolumetric RNA separation solution, centrifuging 16000g of the mixture for 10 minutes, and separating supernate; performing RT-PCR (reverse transcription-polymerase chain reaction); manufacturing a standard curve; and performing calculation to obtain a gene calibration initial copy number Q, and comparing the Q value of the miR-128 and the geometrical mean of Q values of reference gene U6 and miR-16 to obtain a relative expression amount of the minRNA-128. According to the method for testing the minRNA-128 in colorectal cancer serum, important references are provided for early discovery and early treatment of colorectal cancer.

Description

technical field [0001] The invention relates to a special primer, a kit for detecting colorectal cancer serum miRNA-128, and a method for detecting the expression level of colorectal cancer serum miRNA-128, belonging to the technical field of molecular biology detection. Background technique [0002] Colorectal cancer is one of the most common malignant tumors of the digestive system. With the changes in people's lifestyle and dietary structure, the incidence and mortality of colorectal cancer are increasing year by year. Colorectal cancer is relatively easy to treat in the early stages, but the prognosis is often poor in the advanced stages. According to statistics, the 5-year survival rate of colorectal patients without invasion and metastasis can be as high as 90%, the 5-year survival rate of patients with local metastasis is 68%, while the 5-year survival rate of patients with distant metastasis is only 11%. . Therefore, early detection is the key to improve the progno...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王传新张欣王海燕
Owner SHANDONG UNIV QILU HOSPITAL
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