Project description:The purpose was to measure expression changes in human circular RNAs. Total RNA was harvested from KSHV-infected HUVEC and MC116 cells. A portion of the RNA was digested with RNase R to enrich for circular RNAs.
Project description:Viruses subvert macromolecular pathways in the infected host to aid in viral gene amplification or to counteract innate immune responses. Recently, roles for host-encoded non-coding RNAs, such as microRNAs, have been found to encode pro- and anti-viral functions. One class of non-coding RNAs are circular RNAs that are generated by a nuclear back-splicing mechanism of pre-mRNAs. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circular RNAs were up-regulated or down-regulated in infected cells. Identified circular RNAs displayed both pro- and anti-viral effects.
Project description:Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq
Project description:Total RNA of KSHV-infected cells were extracted and RT-PCR was performed using viral circRNA specific divergent primers. Amplicons were sequenced with MiSeq.
