Quantification of siRNA Expression in Transgenic Cotton

Total small RNA population was isolated from the leaves of transgenic and NT control plants^{43 (link)}. It was performed to assess the expression level of siRNA in transgenic (T₀, T₁) plants with respect to corresponding non-transformed cotton plants. The low molecular weight RNA were separated on denaturing polyacrylamide gel (PAGE 15%) and transferred onto a Hybond-N nylon membrane (GE Healthcare, UK). Hybridization was performed with CLCuMuV-C4 gene-based DIG-11-dUTP labelled probe at 42 °C in DIG Easy Hyb solution using DIG DNA labelling and detection kit (Roche Diagnostics, Germany). The membrane was equilibrated in Detection buffer containing NBT/BCIP-T under Gel Doc™ EZ System (BioRad, USA) using the Image Lab™ software Version 4.1. siRNA fragments (20–25 nt long) were visualized, which were compared with the original PAGE exhibiting ultra-low range RNA marker (Thermo Scientific, Germany).