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

Noa Bossel Ben-Moshe, Shlomit Gilad, Gili Perry, Sima Benjamin, Nora Balint-Lahat, Anya Pavlovsky, Sharon Halperin, Barak Markus, Ady Yosepovich, Iris Barshack, Einav Nili Gal-Yam, Eytan Domany, Bella Kaufman, Maya Dadiani

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Article number419
JournalBMC genomics
Volume19
Issue number1
DOIs
StatePublished - May 30 2018

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Gene Expression Profiling
Paraffin
Formaldehyde
Messenger RNA
RNA Sequence Analysis
Transcriptome
Poly A
RNA
Breast Neoplasms
Gene Expression
Costs and Cost Analysis
Genes

Keywords

  • Breast cancer
  • FFPE
  • Poly-a capturing
  • RNA sequencing
  • Ribosomal depletion

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Bossel Ben-Moshe, N., Gilad, S., Perry, G., Benjamin, S., Balint-Lahat, N., Pavlovsky, A., ... Dadiani, M. (2018). mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues. BMC genomics, 19(1), [419]. https://doi.org/10.1186/s12864-018-4761-3

mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues. / Bossel Ben-Moshe, Noa; Gilad, Shlomit; Perry, Gili; Benjamin, Sima; Balint-Lahat, Nora; Pavlovsky, Anya; Halperin, Sharon; Markus, Barak; Yosepovich, Ady; Barshack, Iris; Nili Gal-Yam, Einav; Domany, Eytan; Kaufman, Bella; Dadiani, Maya.

In: BMC genomics, Vol. 19, No. 1, 419, 30.05.2018.

Research output: Contribution to journalArticle

Bossel Ben-Moshe, N, Gilad, S, Perry, G, Benjamin, S, Balint-Lahat, N, Pavlovsky, A, Halperin, S, Markus, B, Yosepovich, A, Barshack, I, Nili Gal-Yam, E, Domany, E, Kaufman, B & Dadiani, M 2018, 'mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues', BMC genomics, vol. 19, no. 1, 419. https://doi.org/10.1186/s12864-018-4761-3
Bossel Ben-Moshe N, Gilad S, Perry G, Benjamin S, Balint-Lahat N, Pavlovsky A et al. mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues. BMC genomics. 2018 May 30;19(1). 419. https://doi.org/10.1186/s12864-018-4761-3
Bossel Ben-Moshe, Noa ; Gilad, Shlomit ; Perry, Gili ; Benjamin, Sima ; Balint-Lahat, Nora ; Pavlovsky, Anya ; Halperin, Sharon ; Markus, Barak ; Yosepovich, Ady ; Barshack, Iris ; Nili Gal-Yam, Einav ; Domany, Eytan ; Kaufman, Bella ; Dadiani, Maya. / mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues. In: BMC genomics. 2018 ; Vol. 19, No. 1.
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AU - Balint-Lahat, Nora

AU - Pavlovsky, Anya

AU - Halperin, Sharon

AU - Markus, Barak

AU - Yosepovich, Ady

AU - Barshack, Iris

AU - Nili Gal-Yam, Einav

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KW - Breast cancer

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KW - Poly-a capturing

KW - RNA sequencing

KW - Ribosomal depletion

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