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A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples

BACKGROUND: Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. It is important to accurately quantitate gene expression with degraded and small amounts of total RNA from FF...

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Autores principales: Lin, Xiaojing, Qiu, Lihong, Song, Xue, Hou, Junyan, Chen, Weizhi, Zhao, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842158/
https://www.ncbi.nlm.nih.gov/pubmed/31703614
http://dx.doi.org/10.1186/s12864-019-6166-3
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author Lin, Xiaojing
Qiu, Lihong
Song, Xue
Hou, Junyan
Chen, Weizhi
Zhao, Jun
author_facet Lin, Xiaojing
Qiu, Lihong
Song, Xue
Hou, Junyan
Chen, Weizhi
Zhao, Jun
author_sort Lin, Xiaojing
collection PubMed
description BACKGROUND: Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. It is important to accurately quantitate gene expression with degraded and small amounts of total RNA from FFPE materials. RESULTS: High concordance in transcript quantifications were shown between FF and FFPE samples using the same kit. The gene expression using the TaKaRa kit showed a difference with other kits, which may be due to the different principle of rRNA depletion or the amount of input total RNA. For seriously degraded RNA from FFPE samples, libraries could be constructed with as low as 50 ng of total RNA, although there was residual rRNA in the libraries. Data analysis with HISAT demonstrated that the unique mapping ratio, percentage of exons in unique mapping reads and number of detected genes decreased along with the decreasing quality of input RNA. CONCLUSIONS: The method of RNA library construction with rRNA depletion can be used for clinical FFPE samples. For degraded and low-input RNA samples, it is still possible to obtain repeatable RNA expression profiling but with a low unique mapping ratio and high residual rRNA.
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spelling pubmed-68421582019-11-14 A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples Lin, Xiaojing Qiu, Lihong Song, Xue Hou, Junyan Chen, Weizhi Zhao, Jun BMC Genomics Methodology Article BACKGROUND: Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. It is important to accurately quantitate gene expression with degraded and small amounts of total RNA from FFPE materials. RESULTS: High concordance in transcript quantifications were shown between FF and FFPE samples using the same kit. The gene expression using the TaKaRa kit showed a difference with other kits, which may be due to the different principle of rRNA depletion or the amount of input total RNA. For seriously degraded RNA from FFPE samples, libraries could be constructed with as low as 50 ng of total RNA, although there was residual rRNA in the libraries. Data analysis with HISAT demonstrated that the unique mapping ratio, percentage of exons in unique mapping reads and number of detected genes decreased along with the decreasing quality of input RNA. CONCLUSIONS: The method of RNA library construction with rRNA depletion can be used for clinical FFPE samples. For degraded and low-input RNA samples, it is still possible to obtain repeatable RNA expression profiling but with a low unique mapping ratio and high residual rRNA. BioMed Central 2019-11-08 /pmc/articles/PMC6842158/ /pubmed/31703614 http://dx.doi.org/10.1186/s12864-019-6166-3 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Lin, Xiaojing
Qiu, Lihong
Song, Xue
Hou, Junyan
Chen, Weizhi
Zhao, Jun
A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title_full A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title_fullStr A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title_full_unstemmed A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title_short A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples
title_sort comparative analysis of rna sequencing methods with ribosome rna depletion for degraded and low-input total rna from formalin-fixed and paraffin-embedded samples
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842158/
https://www.ncbi.nlm.nih.gov/pubmed/31703614
http://dx.doi.org/10.1186/s12864-019-6166-3
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