RNA isolation and Real-time PCR analysis of activation-tagged line

Total RNA from leaf and root tissues of activation-tagged transgenic and wild-type BPT 5204 (both control and stressed conditions) was isolated according to the manufacturer’s instructions (RNAiso Plus, Takara, Japan) and PCR was performed as per Manimaran et al.⁸⁰. Two microgram of total RNA was used for first-strand cDNA synthesis using oligo d(T) primers (SMART MMLV RT, Takara, Japan). The cDNA after RNaseH treatment was mixed with 12.5 µL of 2X KAPA SYBR® FAST qPCR mix universal (KAPA Biosystem, USA), 2 µM each of forward and reverse primers in a final volume of 20 µL. PCR with no template control (NTC) was also performed for each primer pair. The real-time PCR was performed in Mastercycler® ep realplex (Eppendorf, Germany). The conditions for qRT-PCR were as follows. 3 min at 95 °C, 35 cycles of 95 °C - 3 s and 60 °C - 20 s in 96-well optical reaction plates (Axygen, USA). The amplicon specificity was verified by melt curve analysis from 60 to 95 °C after completion of 35 cycles. Each sample was replicated thrice. The comparative threshold cycle (C_T) method was used to quantify the relative expression levels in real-time PCR. OsActin1 was used as a reference gene to normalize gene expression. The ΔC_T for BPT-WT (control condition) was used as a control for the calculation of relative changes in expression in activation-tagged transgenic line (control), WT (NaCl stressed) and transgenic line (NaCl stressed). Relative expression level of genes was calculated using the ΔΔC_TT formula⁸¹. Primers used for gene expression studies and stress-responsive genes (OsP5CS1, OsNAC6, OsLEA3, OsSOS1, OsZIP23, OsNHX1 and OsSalT) are listed in Supplementary Table ¹.