Bhamidimarri, Poorna Manasa;
Salameh, Laila;
Mahdami, Amena;
Abdullah, Hanan Wael;
Mahboub, Bassam;
Hamoudi, Rifat;
(2024)
LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.
Heliyon
, 10
(12)
, Article e32896. 10.1016/j.heliyon.2024.e32896.
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Abstract
Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.
| Type: | Article |
|---|---|
| Title: | LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.heliyon.2024.e32896 |
| Publisher version: | https://doi.org/10.1016/j.heliyon.2024.e32896 |
| Language: | English |
| Additional information: | © The Author(s), 2024. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CC-BY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Keywords: | Linear amplification, Formalin-fixed paraffin embedded, RNA-Seq, Degraded, Transcriptomic analysis |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci > Department of Surgical Biotechnology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10205471 |
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