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An optimised trizol-based protocol for the improvement of rna extraction yield of tomato stem
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Document Title
An optimised trizol-based protocol for the improvement of rna extraction yield of tomato stem
Author
Afifah A., Nounurai P., Ferniah R.S., Kusumaningrum H.P., Wulandari D., Budiharjo A.
Name from Authors Collection
Affiliations
Department of Biology, Faculty of Science and Mathematics, Diponegoro University, Jl. Prof Soedharto SH, Semarang, 50275, Indonesia; Genome Technology Research Unit, National Centre for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Pathumthani, Khlong Luang, 12120, Thailand; Biotechnology Study Program, Diponegoro University, Jl. Prof Soedharto SH, Semarang, 50275, Indonesia; Molecular and Applied Microbiology Laboratory, Central Laboratory of Research and Service, Diponegoro University, Jl. Prof. Sudharto SH, Semarang, 50275, Indonesia; School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand
Type
Article
Source Title
Pertanika Journal of Tropical Agricultural Science
ISSN
15113701
Year
2021
Volume
44
Issue
3
Page
673-684
Open Access
Gold
Publisher
Universiti Putra Malaysia
DOI
10.47836/pjtas.44.3.10
Abstract
One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/ A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications. © 2021 Universiti Putra Malaysia. All rights reserved.
Keyword
Funding Sponsor
Kementerian Pendidikan dan Kebudayaan
License
CC BY-NC-ND
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Publication Source
Scopus
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An optimised trizol-based protocol for the improvement of rna extraction yield of tomato stemDownload