RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification

Abstract

High quality RNA is critical for molecular and viral diagnostic applications. In case of citrus, polyphenol and polysaccharide rich content hinders many RNA isolation methods causing low quality RNA. These shortcomings can affect the Citrus tristeza virus (CTV) detection which is responsible to cause worldwide destruction of citrus orchards. Thus, in this study we analysed the RNA quality, quantity and integrity of different commercial and conventional methods. Commercial methods include TRIzol and kitbased method (PureLink RNA kit), while Cetyl trimethylammonium bromide (CTAB) and SDS/ Phenol (TENS-PCI) based method were optimized as conventional methods. For further validation, amplification potential was evaluated by performing Reverse transcriptase polymerase chain reaction (RT PCR) of CTV coat protein (p25) and mRNA internal control (nad5) genes to affirm RNA quality. Results indicated poor RNA recovery from TRIzol method with no successful amplification at RT PCR. Contrastingly, the modified conventional methods indicated RNA quality and purity for nucleic acids at Absorbance 260/280 (1.8–2.1) and Absorbance 260/230 (1.8–2.2) proving drastic improvement in RNA quality with reproducible amplification results. These results were further demonstrated with stored samples of different citrus varieties which indicated poor RNA integrity but successful RT PCR amplification. Consequently, our results may provide a brief comparison between isolation procedures by providing reference values to determine RNA purity requirements prior to RT PCR and help to determine rapidity, reproducibility and yield for each isolation method.