The Research On A Method Replacing Reverse Transcription To Accurately Detect The Relative Concentration Of Non-coding RNA

After biologists realized that ncRNA should be an ignored key player in life science, identification of enormous ncRNA genes was the main purpose for researchers. Now, thousands of human ncRNAs genes have been identified and sequenced, which are preserved in several databases. A proper method which can precisely determine the stead state level of ncRNAs in human cells or tissue has proven to be a powerful tool for discovering the biological, pathological and clinical roles of ncRNAs in health and disease. Reverse transcription coupled real time PCR had been broadly and most frequently used to measure the steady level of a single ncRNA. However, the efficiency of reverse transcription significantly influences the followed real time PCR detection. So we have established a novel method to replace reverse transcription precisely determining the stead state level of ncRNAs.In this study, we made two short complementary DNA oligos hybridize with the target ncRNA and linked them to obtain a long DNA strand (HL-DNA), which can be applied for followed PCR amplification. To test feasibility of this novel method combined with PCR amplification, the RNU4-1level was measured with gel electrophoresis assay following PCR. Compared with cDNA as the PCR template, HL-DNA generated from same amount of total RNA displayed a higher sensitivity. With the same detection method, the obtained results of a series of prepared samples which contains decreased level of AK026510, a randomly selected ncRNA, were clearly displayed a reduced trend of the AK026510level.Due to HL-DNA as the only DNA template for PCR amplification in the detection system, HL-DNA based qPCR (HL-qPCR) exhibits higher accuracy and repeatability compared with RT-qPCR. With the same primers for PCR amplification, every detected value of a series of samples was more closed to the calculated true value by using HL-qPCR compared with using RT-qPCR.In addition, this method may be applied as an alternative method to measure the activity or concentration of DNA ligase which functions in few DNA repair pathways. And the prelimary studing has confirmed that the concentration of T4DNA ligase can be detected correctly.In summary, this novel HL-qPCR could be applied for the regular ncRNA level detection by replacing RT-qPCR due to its accuracy, repeatability and reproducibility.