Research And Application Of High Throughput RNASeq Method For Micro Sample

The transcriptome is the set of all RNA molecules transcribed under a specific developmental period or functional state in one cell or a population of cells.The transcriptome of a selected organ or tissue in an organism under a specific condition can be obtained quickly and completely via the high-throughput next-generation cDNA sequencing technique.Because of the temporal and spatial specificity of RNA samples,as well as the limited amount of patient tissues,precious samples and single cells,the amount of RNA extracted can be low to ng or even pg level.This study compares routine transcriptome preparation method with three mostly utilized micro scale RNA amplification kit,SMARTer?PCR cDNA Synthesis Kit,Ovation RNASeq System and TransPlex?R Complete Whole Transcriptome Amplification Kit,in order to screen for the best high-throughput sequencing method of micro-scale RNA samples.And the standard and non-standard RNA samples were taken to evaluate the repeatability and stability of the selected method.First,RNA amplification,library construction and sequencing were sequentially performed with 2ng or 10ng RNA reference standard UHRR,andthen sequencing PE90in Hiseq2000 platform.The comparison of gene mapping rates,exon mapping rates,intron mapping rates,intergenic mapping rates,correlation with QPCR,gene coverage rate,and randomnesswith routine transcriptome indicates that amplification with SMARTer?PCR cDNA Synthesis Kit shows the most similar result.Then,we further evaluate and adjust experiment condition,data repeatability and correlation with 2ng,10ng,50ng or 100ng UHRR and HBRR.Then a final evaluation of the method was performed with total RNA sample from human blood cells to confirm that amplification with SMARTer^TM PCR cDNA Synthesis Kit provides the best method for micro-scale RNA sample study.The micro-scale RNA study method we established shows its stability and reliability according to different experiment results,and can be widely applied to eukaryotic micro-scale transcriptomic study.This method can not only solve the micro-scale RNA study problem,provide scientific study reference data,but also promote the development of biomedical science,offer efficient methods for human health study.