Optimization of RNA extraction method from latex of the rubber tree Hevea brasiliensis for gene expression analysis by RT-PCR

Abstract

High-quality nucleic acids (DNA and RNA) are fundamental for genomic/molecular research. Understanding the molecular mechanisms of rubber biosynthesis and its regulation relies on obtaining pure RNA (ribonucleic acid) for gene expression analysis. Mature leaves or limited leaf samples often present challenges in extracting high quality RNA. This study presents a precise, efficient, and reliable method for extracting pure, high-quality RNA from the latex of the rubber tree (Hevea brasiliensis) for gene expression analysis. Traditional methods using Trizol reagent, chloroform, or salts can introduce contaminants such as excess phenol and proteins from the latex, compromising the quality of RNA. To address this, an additional chloroform extraction step, multiple RNA washes with 95% ethanol, and a freeze-drying the RNA pellet at -20°C overnight was incorporated. The added chloroform step reduced RNA contamination with phenol or proteins from the interphase of solvents. Repeated washes with 95% ethanol effectively eliminated residual salts from the isopropanol RNA precipitation step and any remaining phenol and chloroform, further enhancing RNA purity (checked by Nano-spectrophotometer). The freezer-drying step mitigates RNA degradation due to temperature fluctuations in the working area. These modifications significantly improve the purity, quality, accuracy, and reliability of RNA quantification (the concentration and purity). The efficacy of the modified RNA extraction protocol was validated by assessing the quality, quantity and purity of the isolated RNA using Nano spectrophotometric determination (A260/A280 ratio was around 2.0 and A260/A230 ratio was around 2.0 to 2.2) and 2% agarose gel electrophoresis. The downstream applicability of the isolated RNA was further validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) for gene expression analysis of drought tolerance and ref gene. The study proved the suitability of a modified RNA extraction protocol to isolate RNA from rubber latex to generate accurate qRT-PCR results.