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●第8回日本RNAi研究会 第3回日本細胞外小胞学会JSEVにてランチョンセミナーを行いました。

演題 次世代シーケンサー(NGS)とqPCRプラットフォームによるB型およびC型慢性肝炎患者血清を用いたmicroRNAプロファイル解析データのご紹介
演者 マイケル・ハンセン【エキシコン社】
開催日 2016年9月2日(金)12:00〜13:00
microRNAs represent the most well described class of small RNAs (21-23nt) and have been shown to function as post-transcriptional regulators of gene expression. The high relative stability of microRNA in common clinical source materials (e.g. FFPE blocks, as well as biofluids such as plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNA quantification as promising new molecular biomarkers for a wide range of applications. Furthermore microRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology.
Improved non-invasive biomarkers are needed to manage patients with chronic HBV and HCV infections, and serum microRNAs may represent a new class of biomarkers due to the important role that microRNAs play in the interaction between virus and host (Fan & Tang, 2014). In a previous qPCR microRNA profiling study, liver-derived microRNAs were found to be detected at high levels in the sera of HBV patients, and a microRNA signature associated was discovered that could help identify patients with both natural and therapy induced immune control of chronic HBV infection (Brunetto et al., 2014).
Profiling microRNAs in biofluids is challenging due to the limited amount of RNA present in biofluids, as well as presence of inhibitory compounds which have the potential to inhibit downstream enzymatic processes. In this study we compare Next Generation Sequencing (NGS) technology with qPCR-based profiling of microRNAs in biofluids. We have optimized each step of the workflow from RNA isolation to RNA QC and NGS library preparation or RT-qPCR in order to obtain reliable results from challenging biofluid samples.
Genome-wide microRNA profiling was performed at Exiqon Services on baseline serum samples from the same patients with either chronic HBV infection or chronic HCV infection (n=5 patients per group), using both NGS (Illumina NextSeq500) and qPCR (miRCURY LNA Universal RT microRNA PCR System, Human Panels I+II V3) technologies, following protocols optimized for biofluids. Overall good agreement was obtained between NGS and qPCR technologies for biofluid microRNA profiling, when following the protocols optimized for biofluids. However, some platform differences were observed, highlighting the importance of careful validation.
While similar numbers of microRNAs could be identified in the serum of HBV and HCV patients, on average a higher percentage of microRNA reads were identified in the HBV serum samples compared to the HCV serum samples. This difference is attributed to the large amount of liver-derived microRNA present in the serum of HBV patients. Consistent with this, certain liver-derived microRNAs (miR-122-5p, miR-192-5p, miR-99a-5p), were found to be detected at lower levels in serum from HCV patients compared to HBV patients.

Fan & Tang. Complex interactions between microRNAs and hepatitis B/C viruses. World J Gastroenterol. 2014 Oct 7;20(37): 13477-92.
Brunetto et al., A serum microRNA signature is associated with the immune control of chronic hepatitis B virus infection. PLoS One. 2014 Oct 28;9(10): e110782.

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