Molecular marker for early pancreatic neoplasm detection as well as detection method and application thereof
A molecular marker, pancreatic tumor technology, applied in the field of biotechnology and clinical molecular diagnostic drug development, can solve the problems of difficult early diagnosis of pancreatic cancer, low sensitivity and specificity of biomarkers, etc.
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Embodiment 1
[0075] Early screening of miRNAs and construction of precise drug detection system (SOP)
[0076] S1. To screen pancreatic cancer miRNAs, design specific probes and primers for target miRNAs according to the principle of TaqMan probe technology; react target miRNAs in U6 in the same system; the designed primers and probes are as follows:
[0077] Table 1. Design of primers and probe sequences for target miRNAs
[0078]
[0079]
[0080] S2.miRNA extraction technology optimization
[0081] (1) Determine the extraction reagent. Screened three commonly used commercial RNA isolation kits, namely TRIzol (Ambion), TRIzol LS Reagent (Invitrogen) and miRNeasy Serum / Plasma kit (Qiagen), compared the quality of the extracted miRNA, and evaluated the cost and ease of operation degree and other factors, select the extraction reagent with the best comprehensive performance. The three extraction reagents were used to extract the same test sample (taking Sw1990 pancreatic cancer cel...
Embodiment 2
[0096] Utilizing the multiple fluorescent probe detection technology for miRNA in serum established by the present invention, miR-30c, miR-24, miR-23a, miR-132, miR-21, let The expression of -7i, miR-1207, miR-130, miR-200c, miR-154, and miR-57 were divided into 6 combinations, namely,
[0097] Combination 1: miR-24 / miR-23a / miR-132 / miR-30c;
[0098] Combination 2: miR-130 / miR-200c / miR-154 / miR-30c;
[0099] Combination 3: miR-24 / miR-132 / miR-1207 / let-7i;
[0100] Combination 4: miR-30c / miR-24 / miR-59 / miR-132;
[0101] Combination 5: miR-130 / miR-21 / let-7i / miR-30c;
[0102] Combination 6: miR-30c / miR-154 / miR-23a / miR-57.
[0103] Specifically: serum samples from patients from Shanghai Renji Hospital, Dalian Medical University, Peking Union Medical College Hospital and Nanjing Medical University (including early pancreatic ductal adenocarcinoma, pancreatitis, pancreatic ductal papillary mucinous tumor) and normal samples from Hunan Xiangya Hospital Human serum samples are operat...
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