MicroRNA/Small RNA Profiling Using Frozen and Paraffin Renal Cell Carcinoma Tissue Cohort by Next-Generation Sequencing
L Weng, X Wu, H Gao, B Mu, X Li, J Wang, C Guo, J Jin, M Covarrubias, L Weiss, H Wu. City of Hope, Duarte, CA
Background: MicroRNA (miRNA) is a group of small non-coding regulating RNAs and has shown altered expression in cancer. Until recently, microarray and real-time PCR have been widely used for quantitative miRNA studies. With the major limitations in test specificity and capability of detecting unknown miRNA and other small RNA targets, they are being replaced by the newly developed next-generation sequencing technology. Although next-generation sequencing has been recently used to study miRNA/small RNA expression in cancer primary cell culture or cell lines and shown great potential, similar studies applied to human cancer tissue cohorts have not been reported. Specifically, FFPE samples have not been used for small RNA studies by next-generation sequencing, since it has been assumed to have insufficient quality of RNA. However, clinical tissue samples are routinely stored as FFPE samples. Therefore, establishing a method to use FFPE samples for miRNA expression profiling by deep sequencing would make future clinical cohort/trial-based studies possible.
Design: (1) A clear cell renal cell carcinoma (CCRCC) cohort with both frozen and corresponding FFPE samples was established. It included frozen tissue from 3 benign kidney and 3 CCRCC samples, and their FFPE counterparts. (2) Deep sequencing of small RNA in all samples using Illumina GAII was performed. (3) Microarray testing on all frozen samples was performed using an Agilent MicroRNA Array. (4) The miRNA expression level of each sample was compared between deep sequencing and microarray platforms. (5) miRNA/small RNA expression was profiled in both frozen and FFPE cohort sets and the results were compared.
Results: (1) miRNAs and other small RNAs can be detected by deep sequencing in frozen samples, and the expression level of miRNAs is highly correlated with that measured by microarray. (2) Differentially expressed miRNAs identified in deep sequencing highly correlated with the microarray and with real-time PCR results. (3) miRNA and other small RNAs can be detected by deep sequencing in FFPE samples, and the expression levels of miRNA are highly correlated between frozen and FFPE sample pairs. (4) Differentially expressed miRNAs identified in FFPE and frozen samples with deep sequencing are highly similar.
Conclusions: (1) Deep sequencing platform can be used for human tissue cohort studies to profile miRNA/small RNA expression in cancer. (2) A FFPE tissue cohort can be used reliably to profile miRNA expression in cancer.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Tuesday, March 23, 2010 9:00 AM
Platform Session: Section H 1, Tuesday Morning