mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

BMC Genomics

Gili Perry¹  Sharon Halperin¹  Maya Dadiani¹  Einav Nili Gal-Yam²  Bella Kaufman²  Iris Barshack³  Ady Yosepovich³  Nora Balint-Lahat³  Anya Pavlovsky³  Eytan Domany⁴  Noa Bossel Ben-Moshe⁴  Barak Markus⁵  Shlomit Gilad⁵  Sima Benjamin⁵ 

[1] Chaim Sheba Medical Center, Cancer Research Center;Chaim Sheba Medical Center, Institute of Oncology;Chaim Sheba Medical Center, Institute of Pathology;Department of Physics of Complex Systems, Weizmann Institute of Science;The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science;

关键词: RNA sequencing;    FFPE;    Poly-a capturing;    Ribosomal depletion;    Breast cancer;   

DOI  :  10.1186/s12864-018-4761-3

来源: DOAJ

【 摘 要 】

Abstract Background The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3′-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2–3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

【 授权许可】

Unknown