Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seqReport as inadecuate




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BMC Genomics

, 15:1087

Human and rodent genomics

Abstract

BackgroundFormalin-fixed paraffin-embedded FFPE tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing NGS is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.

ResultsWe optimized strategies for whole transcriptome sequencing RNA-seq using FFPE tissue. Ribosomal RNA rRNA was successfully depleted by competitive hybridization using the Ribo-zero™ Kit Epicentre Biotechnologies, and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen FF tissue and quantitative PCR qPCR. Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs fragments per kilobase of exon per million fragments mapped and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma RCC correlated highly r = 0.919 for tumor 1 and r = 0.954 for tumor 2. On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2. Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors tumor 1-tumor 2 correlated well when comparing qPCR and RNA-seq r = 0.890. Expression fold changes between tumors from different risk groups our high risk RCC-The Cancer Genome Atlas, TCGA, low risk RCC also correlated well when comparing RNA-seq from FF and FFPE tumors r = 0.887.

ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.

KeywordsFormalin-fixed paraffin-embedded FFPE qPCR Renal cell carcinoma RCC RNA-seq Gene expression Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-15-1087 contains supplementary material, which is available to authorized users.

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Author: Ping Li - Andrew Conley - Hao Zhang - Hyung L Kim

Source: https://link.springer.com/







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