Development of a rapid and modified total RNA extraction method from polyphenolic-rich Gossypium hirsutum

RNA extraction from polyphenolic-rich plants poses significant challenges, demanding precise sample handling and rigorous experimental conditions to ensure high yield and quality of the RNA. Numerous kits are available for RNA extraction from various sources, however extracting RNA from phenolic-rich plants requires special attention. On the other hand, manual extraction methods are time-consuming and involve harsh chemicals (LiCl, phenol, guanidine thiocyanate). To address this issue, we have improvised and compared three strategies for RNA extraction. We found that CTAB-based extraction buffer produces high-quality RNA from cotton, followed by ammonium acetate purification, within two hours. Column purification (DirectZol RNA purification kit) followed by CTAB extraction step not only accelerates the RNA purification process (∼30 min) but produces high-quality RNA suitable for various downstream applications such as real-time quantitative PCR, sequencing, and RNA library preparation. The RNA yield ranged between 80-100µg/100mg for CTAB-ammonium acetate as compared to up to 12µg/100mg for guanidium thiocyanate-ammonium acetate-based extraction from cotyledonary leaves of cotton. RNA purification from mature cotton leaves was unsuccessful with guanidinium thiocyanate. The downstream applications like qPCR and the sequencing results depicted the contaminant-free RNA from phenolic-rich mature cotton leaves. The modified method is rapid and could be completed within 2 hours for completely manual procedure For extra fast purification CTAB-buffer extraction can be combined with RNA purification kit (∼30-40 min) The modified method yields high quality and phenol-free RNA for downstream applications like real time quantitative PCR, sequencing, and RNA library preparation.