A modified SDS-LiCl method for high quality RNA isolation from mucilage rich okra Tissues and downstream virus...

Okra (Abelmoschus esculentus L.) is an economically important vegetable crop and an important host of several viral pathogens, including tobacco streak virus (TSV). Reliable molecular diagnostics and gene expression studies require high-quality RNA; however, RNA isolation from okra is particularly challenging due to the abundance of mucilage, polysaccharides, and phenolic compounds that co-extract with nucleic acids and interfere with downstream applications. In the present study, a modified SDS-LiCl RNA extraction method was developed to effectively remove mucilage and obtain high-quality RNA. Its performance was compared with three existing protocols, CTAB-LiCl, TRIzol, and the RNeasy Plant Mini Kit for RNA isolation from five different okra tissues, namely leaf, stem, flower, fruit, and seed. RNA quality and quantity were evaluated using spectrophotometric measurements and agarose gel electrophoresis, while suitability for downstream applications was assessed through RT-PCR and RT-qPCR-based detection of TSV. Among the methods evaluated, the modified SDS-LiCl protocol consistently produced improved RNA yield, purity, and integrity across all tissue types. The modified protocol incorporated higher NaCl and SDS concentrations, PVP and β-mercaptoethanol supplementation, additional chloroform:isoamyl alcohol purification, and selective LiCl-sodium acetate precipitation to effectively remove mucilage, polysaccharides, phenolic compounds, and other PCR inhibitors. Among all the methods modified SDS-LiCl and RNeasy Plant Mini Kit yielded mucilage -free RNA. Principal Component Analysis further identified the modified SDS-LiCl method provided the best balance between RNA quality and recovery. RT-PCR detection of the TSV coat protein gene was 100% for RNA obtained using both the modified SDS-LiCl protocol and the RNeasy Plant Mini Kit, compared with 80% for CTAB-LiCl and 70% for TRIzol methods, respectively. RT-qPCR analysis showed improved amplification performance for SDS-LiCl-extracted RNA, with the lowest Ct value for the endogenous elongation factor gene (15.33). The modified SDS-LiCl protocol exhibited the highest analytical sensitivity, enabling TSV detection up to the 10⁻⁴ dilution, one order of magnitude greater than CTAB-LiCl and two orders of magnitude greater than TRIzol and the RNeasy Plant Mini Kit. Overall, the modified SDS-LiCl protocol provides a reliable, cost-effective, and highly efficient strategy for isolation of high-quality RNA from mucilage-rich okra tissues and offers a valuable tool for virus diagnostics, gene expression studies, and other molecular applications in okra and related crops.