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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9164037 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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