The Multiple RT-qPCR System Establishment For Early Diagnosis Of Pancreatic Cancer

Pancreatic cancer(PC)is a cancer with insidious onset and extremely high malignancy,with a survival rate as low as 5%,accounting for about 4.5% of cancer-related deaths worldwide.It is expected to be one of the leading causes of cancer-related deaths by 2030,second only to lung cancer.According to clinical statistics,about 77% of PC patients will die within one year after diagnosis,and 94% of patients will die within five years.Numerous studies have shown that microRNA(miRNA)is abnormally expressed in the serum of various cancer patients,which is either high or low,so it can be used as a new tumor biomarker.Efficient detection of miRNA in serum can not only provide early diagnosis for pancreatic cancer,improve the survival rate of patients,but also provide the reliable basis for judging the development process and prognosis of pancreatic cancer.This study through consulting a large number of literatures,collecting and sorting out miRNA which have been confirmed that are stability existence and abnormal expression in the pancreatic cancer,then use the pancreatic cancer cell line Sw1990 to verify the expression level of 30 miRNA,finally screen the following 4 miRNA as target testing gene,such as miR-24-1-5p* ? miR-181a-5p ? miR-10b-5p and miR-132-3p.Experimental core parts are mainly to optimize the extract serum microRNAs technology and establish the multiple RT-qPCR(mRT-qPCR)system.First,TRIzol LS,the best comprehensive efficiency,was selected from three commonly used miRNA extraction reagents,and its extraction process was optimized to finally determine the extraction scheme.Secondly,conditions were explored and optimized for multiple factors affecting the amplification efficiency of multiple rt-qpcr.Finally,in the reverse transcription process,the optimal RNA loading amount was 50 ng,the dNTP mixture dosage was 0.15?L,and the Multiscribe RT enzyme dosage was 1.0?L.In the PCR amplification process,the amount of dNTPs mixture was 2.5 ?L and the amount of primers and probes was 2.0 ?L per 20 ?L system.Finally,Taqman probe sequences of four target mirnas were optimized.Above all,mRT-qPCR was constructed.