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Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?

RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits...

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Autores principales: Hilmi, Marc, Armenoult, Lucile, Ayadi, Mira, Nicolle, Rémy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9164037/
https://www.ncbi.nlm.nih.gov/pubmed/35678677
http://dx.doi.org/10.3390/cimb44050148
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author Hilmi, Marc
Armenoult, Lucile
Ayadi, Mira
Nicolle, Rémy
author_facet Hilmi, Marc
Armenoult, Lucile
Ayadi, Mira
Nicolle, Rémy
author_sort Hilmi, Marc
collection PubMed
description RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits are proposed for FFPE samples yet no comparison on low quantities were performed. To select the most reliable, cost-effective, and relevant RNA-Seq approach, we applied five FFPE-compatible kits (based on 3′ capture, exome-capture and ribodepletion approaches) using 8 ng to 400 ng of FFPE-derived RNA and compared them to Nanostring on FFPE samples and to a reference PolyA (Truseq) approach on flash-frozen samples of the same tumors. We compared gene expression correlations and reproducibility. The Smarter Pico V3 ribodepletion approach appeared systematically the most comparable to Nanostring and Truseq (p < 0.001) and was a highly reproducible technique. In comparison with exome-capture and 3′ kits, the Smarter appeared more comparable to Truseq (p < 0.001). Overall, our results suggest that the Smarter is the most robust RNA-Seq technique to study small FFPE samples and 3′ Lexogen presents an interesting quality–price ratio for samples with less limiting quantities.
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spelling pubmed-91640372022-06-04 Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used? Hilmi, Marc Armenoult, Lucile Ayadi, Mira Nicolle, Rémy Curr Issues Mol Biol Communication RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits are proposed for FFPE samples yet no comparison on low quantities were performed. To select the most reliable, cost-effective, and relevant RNA-Seq approach, we applied five FFPE-compatible kits (based on 3′ capture, exome-capture and ribodepletion approaches) using 8 ng to 400 ng of FFPE-derived RNA and compared them to Nanostring on FFPE samples and to a reference PolyA (Truseq) approach on flash-frozen samples of the same tumors. We compared gene expression correlations and reproducibility. The Smarter Pico V3 ribodepletion approach appeared systematically the most comparable to Nanostring and Truseq (p < 0.001) and was a highly reproducible technique. In comparison with exome-capture and 3′ kits, the Smarter appeared more comparable to Truseq (p < 0.001). Overall, our results suggest that the Smarter is the most robust RNA-Seq technique to study small FFPE samples and 3′ Lexogen presents an interesting quality–price ratio for samples with less limiting quantities. MDPI 2022-05-13 /pmc/articles/PMC9164037/ /pubmed/35678677 http://dx.doi.org/10.3390/cimb44050148 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Hilmi, Marc
Armenoult, Lucile
Ayadi, Mira
Nicolle, Rémy
Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title_full Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title_fullStr Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title_full_unstemmed Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title_short Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
title_sort whole-transcriptome profiling on small ffpe samples: which sequencing kit should be used?
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9164037/
https://www.ncbi.nlm.nih.gov/pubmed/35678677
http://dx.doi.org/10.3390/cimb44050148
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