MicroRNA Analysis Using RNA Extracted from Matched Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh Frozen Samples on SOLiD™ System

摘要

Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. MicroRNAs (miRNAs) regulate gene expression at post-transcriptional level and are considered as important regulators of cancer progression. Next generation sequencing technologies such as SOLiD™ provide an ideal method for measuring the abundance of miRNA molecules in different cancer stages and provide insightful information on tumorigenesis. However, currently there is no good method to systematically study miRNA expression in FFPE samples on next generation sequencing platforms. We have designed and developed a ligation-based miRNA detection method to capture small RNA sequences in FFPE samples and convert them into templates suitable for sequencing on the SOLiD™ System. Total RNA was isolated from matched lung adenocarcinoma FFPE and snap frozen tissues using an Ambion RecoverAll™ kit. A PureLink™ miRNA Isolation kit was used to enrich the small RNA fraction in these total RNA samples. Library preparation using a SOLiD™ Total RNA-Seq kit with modified protocol was performed on the enriched RNA followed by sequencing on SOLiD™ system. Our results show that small RNA extracted from FFPE samples was successfully converted to small RNA libraries. Very similar mapping statistics were obtained from matched FFPE and fresh-frozen samples after SOLiD™ sequencing. A good correlation of miRNA expression pattern was also observed. This suggests that miRNA molecules are less affected by sample degradation and RNA-protein crosslink. This study provides a foundation for miRNA expression analysis on SOLiD™ system using FFPE samples in cancer and other diseases.