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Pertanika Journal of Tropical Agricultural Science

Official journal of Universiti Putra Malaysia for peer-reviewed research across tropical agriculture and allied agricultural sciences.

e-ISSN 2231-8542 ISSN 1511-3701
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Research article

An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

Anis Afifah, Prachumporn Nounurai, Rejeki Siti Ferniah, Hermin Pancasakti Kusumaningrum, Dyah Wulandari and Anto Budiharjo

Volume 44, Issue 3 August 2021 Published 30 August 2021
https://doi.org/10.47836/pjtas.44.3.10
KeywordsRNA extraction, RT-PCR, Solanum lycopersicum, TRIzol
Article content

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.
Supporting literature

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