Abstract
RNA extraction and analyses from tissues using bulk RNA-Sequencing (RNA-Seq) provide a more accurate picture of the gene expression compared to other molecular biology techniques for RNA quantification. Challenges associated with high-quality RNA extraction from skeletal muscles require a modification of standard protocols. Here, we describe a procedure for high-quality RNA isolation from intrinsic laryngeal muscles transferable to skeletal muscles with comparable technical and biological difficulties. Standard protocols for RNA isolation were optimized by maximizing the pooling strategy, determining the sample weight, applying cryogenic muscle disruption, and incorporating RNase-inhibiting reagents during the tissue preparation steps.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tavares L, Alves PM, Ferreira RB, Santos CN (2011) Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma. BMC Research Notes 1:140
Ali N, Rampazzo RCP, Costa ADT et al (2017) Current nucleic acid extraction methods and their implications to point-of-care diagnostics. Biomed Res Int:9306564
Rao MS, Van Vleet TR, Ciurlionis R et al (2019) Comparison of RNA-Seq and microarray gene expression platforms for the toxicogenomic evaluation of liver from short-term rat toxicity studies. Front Genetics 9:636
Puchta M, Boczkowska M, Groszyk J (2020) Low RIN Value for RNA-Seq library construction from long-term stored seeds: a case study of barley seeds. Genes (Basel) 11(10):1190
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63
Mariani A, Bonfio C, Johnson CM et al (2018) pH-driven RNA strand separation under prebiotically plausible conditions. Biochemistry 57(45):6382–6386
Opitz L, Salinas-Riester G, Grade M et al (2010) Impact of RNA degradation on gene expression profiling. BMC Medical Genomics 3(1):36
Sampaio-Silva F, Magalhães T, Carvalho F et al (2013) Profiling of RNA degradation for estimation of post mortem [corrected] interval. PLoS One 8(2):e56507
Jain C (2012) Novel role for RNase PH in the degradation of structured RNA. J. Bacteriol. 194(15):3883–3890
Kemfack AM, Hernandez-Morato I, Moayedi Y et al (2022) An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model. Sci Rep 12(1):21665
Rajkumar AP, Qvist P, Lazarus R et al (2015) Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq. BMC Genomics 16(1):548
Acknowledgments
The authors wish to thank the Pitman and Moayedi labs for contributing their expertise in the development of the present protocol. We also acknowledge Christine Colasacco, Emily Honzel, Almudena Bosch, Midini Annavajhala, and Larissa Williams.
Sources of Funding
This work was supported by the National Institutes of Health grant (1R01DC018060).
Conflicts of Interest
The authors have no personal or institutional interest concerning the authorship and/or publication of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Hernandez-Morato, I., Kemfack, A.M. (2024). Next-Generation Sequencing Application: A Systematic Approach for High-Quality RNA Isolation from Skeletal Muscles. In: Astatke, M. (eds) RNA Amplification and Analysis. Methods in Molecular Biology, vol 2822. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3918-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3918-4_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3917-7
Online ISBN: 978-1-0716-3918-4
eBook Packages: Springer Protocols