Terminal Deoxyribonucleotidyl Transferase-mediated Reverse Transcription PCR For Liver Injury Marker MicroRNA Detection

Micro RNAs have recently emerged as a class of significant disease markers.Realtime fluorescent quantitative reverse transcription PCR(RT-qPCR)is one of the most widely used miRNA detection technologies.However,due to the small size,low content and various species of mi RNA,hybridization forces between primers and probes of PCR required to be stabilized by complex design or chemical modification.In order to solve the above problems,the terminal deoxynucleotidyl transferase(TdT)was introduced into the real-time fluorescence quantitative reverse transcription PCR system.We found that TdT could catalyze the addition of four dNTPs onto the 3’-hydroxy terminus(3’-OH)of RNA to produce DNA sequences.By utilizing this feature,we used TdT to elongate miRNA and further combined reverse transcription PCR to achieve sensitive and selective detection of miRNA without complex probe modification.The details are as follow:RNA-initiated TdT polymerization was characterized by agarose gel electrophoresis and fluorescent labeling denaturing PAGE.The results showed that TdT could use RNA as a trigger and deoxyribonucleotides(d NTPs)as substrates to synthesize DNA.Different types of dNTPs have distinct TDT extension efficiencies.A novel mi RNA detection method has been developed through the combination of RNA-primed TdT polymerization and RT-qPCR.In the proposed method,we firstly applied Td T to extend mi RNAs,and then used the arbitrary primer and the specific primer to amplify the elongated products.The results showed that the sensitivity of this strategy for detection mircro RNA-122(mi R-122)is down to 200 zmol with good specificity.This method was used to study the relationship between liver injury and miR-122 level in serum.We verified that the acetaminophen overexpose leading to the up-regulation of the liver injury biomarker mi R-122.In addition,the TdT-mediated reverse transcription PCR method using each kind of dNTP as a substrate was designed to further explore the utilization of four kinds of dNTPs by TdT in RNA-priming systems.This TdT-mediated reverse transcription PCR method was also applied to nucleic acid quantification.In summary,the RNA priming properties of terminal transferase were investigated.Terminal transferase-mediated reverse transcription PCR method was successfully constructed and applied to the analysis of liver injury markers.This research has enormous potential value in the application of TdT and detection of disease markers.