Establishing a procedure for detecting miRna-425-5p in human plasma using the RT-qPCR methodology

Tóm tắt

Quantitative reverse transcription PCR (RT-qPCR) paired with stem-loop was utilized in this study to establish a process for detecting miRNA-425-5p in human  plasma, contributing to accurate prognosis and early diagnosis of genetic diseases in humans, especially cancer diseases. Blood samples (n=12) collected from healthy volunteers in Ho Chi Minh City, were subjected to plasma extraction and then microRNA (miRNAs) isolation using the TRIzol method. MiRBase, Primer-BLAST, and OligoAnalyzer were used to design and analyze the primers: stem-loop, and miRNA-425-5p sequence-specific primers in two-step RT-qPCR, reverse transcription (RT), and qPCR. The extracted miRNA samples were processed using SensiFAST^TM cDNA KIT (Bioline) in reverse transcription (RT) reaction, and SensiFAST^TM HRM KIT (Bioline) for the qPCR. The RT-qPCR was optimized and improved through sensitivity, stability, and specificity after performing on miRNA samples.The designed stem-loop, forward, and reverse primers have corrected pairing with the miRNA-425-5p, but have an unwanted product of 918 nucleotides in length. Performing the miR-425-5p detection procedure by the RT-qPCR method achieved the following evaluation criteria: specificity (p-value=0.7>0.05), stability (%CV<4%), and sensitivity (99ng/µL).The study successfully established the RT-qPCR procedure for detecting miRNA-425-5p in human plasma.