Optimization of RNA Extraction Protocol for Rat Skeletal Muscle Samples
Journal of Applied Life Sciences International,
Page 10-16
DOI:
10.9734/jalsi/2023/v26i1592
Abstract
Aims: The present study aimed to stablish and characterize an optimized protocol conformation to obtain adequate RNA quality from rodents skeletal muscle samples for sequencing studies.
Place and Duration of Study: The in vivo experiments and analyses were performed in the Laboratory of Biochemistry and Gene Expression – LABIEX of the Superior Institute of Biomedical Science – ISCB from the State University of Ceará - UECE. Between 2017-2020.
Methodology: Were used 23 samples from male Wistar rat skeletal muscle, specifically from soleus muscle. Total RNA extraction was performed using the classic TRIzol® method and commercial kit, merging steps from both. Capillary electrophoresis in the Bioanalyzer platform was used for RNA quality evaluation.
Results: (C) Analyzes of adapted protocol RNA concentration, RIN and rate 28S/18S showed satisfactory results. 28S/18S Ribosomal bands appear well defined, without small traces, which indicates RNA with high integrity and without contamination of genomic DNA.
Conclusion: Obtained RNA quality and integrity data satisfied the exigencies for posterior RNA-seq.
Keywords:
- Sequencing
- RNA extraction
- soleus muscle
How to Cite
References
Bitencourt GA. Chiari L. Valle CB. Laura VA. I live JR. Research and Development Bulletin 29: Evaluation of different total RNA extraction methods from brachiaria leaves and roots. Embrapa Beef Cattle Campo Grande, MS. 2011;1:26.
Available:https://www.infoteca.cnptia.embrapa.br/bitstream/doc/920024/1/BP29.pdf
Accessed 26 March 2018
Zimmermann MT. The importance of biologic knowledge and gene expression context for genomic data interpretation. Frontiers in Genetics. 2018;9:670.
Wang Z, Gerstein M, Snyder, M. RNA-Seq: A revolutionary tool for transcriptomics. Nature Reviews Genetics. 2009;10(1):57-63.
Belesini AA. Transcriptome analysis of sugarcane leaves subjected to prolonged water stress using RNA-Seq. UNESP. 2015:1-140.
Available:https://repositorio.unesp.br/bitstream/handle/11449/134008/000855233.pdf?sequence=1&isAllowed=y
Accessed 26 March 2018
Geraci F, Saha I, Bianchini M. RNA-Seq analysis: Methods, applications and challenges. Frontiers in Genetics. 2020;11:220.
Schroeder A, et al. The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. 2006;7(1):1-14.
Gayral P, Weinert L, Chiari Y, Tsagkogeorga G, Ballenghien M, Galtier N. Next-generation sequencing of transcriptomes: A guide to RNA isolation in nonmodel animals. Molecular Ecology Resources. 2011;11(4):650-661.
Chiari Y, Galtier N. RNA extraction from sauropsids blood: evaluation and improvement of methods. Amphibia-Reptilia. 2011;32(1):136-139.
Marchesi JAP, Santos Silva DB, Borges LJ, Stafuzza NB, Munari DP. Study of genetic variability applied to animal genomics. Special Topics in Applied Genetics, 3rd ed. FUNESP; 2016.
Sambrook JE, Russell DW. Molecular cloning: A laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2002.
-
Abstract View: 110 times
PDF Download: 28 times
