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

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.

Emerging Extraction and Diagnostic Tools for Detection of Plant Pathogens: Recent Trends, Challenges, and Future Scope

“…The TRIzol method is relatively easy and is economically feasible, but the drawback is that due to the presence of phenol and guanidinium isothiocyanate, a low quality of RNA is extracted. When the TRIzol (Invitrogen) process was optimized, it showed enhancement in RNA yield and purity of the tomato stem sample . Another example includes isolation of potato spindle tuber viroid RNA using TRIzol reagent with β-marceptanol .…”

“…When the TRIzol (Invitrogen) process was optimized, it showed enhancement in RNA yield and purity of the tomato stem sample. 27 Another example includes isolation of potato spindle tuber viroid RNA using TRIzol reagent with βmarceptanol. 28 Similarly, in the case of sweet potato feathery mottle virus affecting Chrysanthemum morifolium, TRIzol reagent was used to isolate RNA.…”

Exploring the Role of Microbial Live Factories in Post-Harvest Management of Potatoes-Possible Solution to the Optimization of Supply Chain

“…As averse to senescent sweetening (natural irreversible process occurring as a result of cellular breakdown due to tuber aging); cold-induced sweetening can be partially reversed by temperature reconditioning [37]. Since cold is also an abiotic stress; as an adaptive behavior of tubers to cold stress, it can induce enhanced starch-sugar metabolism and cause sugar accumulation [38]. The contents and basal metabolism of small-molecule metabolites such as sugars and free amino acids directly affect tuber adaptability to cold.…”

“…In a recent study, cold storage significantly affected certain metabolites like sugars, sugar alcohols, amino acids, and organic acids [43]. Based on this, it has been indicated that metabolites such as glucose, fructose, sucrose, asparagine, glutamine, citrate, malate, proline, and 4-aminobutyrate can be potentially utilized for the selection and development of potato cultivars for long-term storage, nutritional, and processing attributes [37][38][39][40]43].…”