RNA Isolation and Quality Evaluation

Early log-phase promastigotes (1 × 10⁸) of both K133WT and K133AS-R were used to isolate total RNA using TRIzol reagent according to the manufacturer’s instruction. Extracted RNA was cleaned up using a RNeasy Plus mini kit (Qiagen, Hilden, Germany). The absorbance of purified RNA was taken at 260 and 280 nm using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The quality and integrity of RNA were assessed on an RNA 6000 Nano Assay Chips on Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA of good quality based on the 260/280 values (Nanodrop, Thermo Scientific, Waltham, MA, USA), rRNA 28S/18S ratios, and RNA integrity number (RIN) was used for further analysis [24 (link)].