Nanodrop lite uv visible spectrophotometer

Total RNA isolation was performed by using Spectrum^TM Plant Total RNA Isolation Kit (Sigma Aldrich, St. Louis, MO, USA) following the manufacturer’s protocol. The quality and the quantity of the total isolated RNA was checked on agarose gel electrophoresis and Nanodrop Lite UV–Visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was used to synthesize cDNA using iScript^TM cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer protocol. The primers for the real-time quantitative qRT-PCR were designed by QuantPrime online tool (https://quantprime.mpimp-golm.mpg.de/). The qRT-PCR was performed in 10 µL PCR mixture (5 µL SYBRGreen, 0.3 µL forward and reverse primer each, 1 µL cDNA (5 ng/µL), 3.4 µL ddH₂O). The house-keeping gene Ubiquitin was used as a reference to normalize the expression values of target genes. The relative expression calculations were performed using the 2^−ΔΔCt formula [43 (link)] for three biological and technical replicates each.