Fast sybr green pcr master mix

Real time-PCR was performed in 20 μl for a set of selected genes using Fast SYBR Green PCR Master Mix (Agilent Technologies, USA). The list of selected genes and oligonucleotide primers (Sigma-Aldrich, USA) used for each gene are listed in Table S1. oligonucleotide primers for vetiver ubiquitin gene were used as the internal control for establishing equal amounts of cDNA in all reactions. The reactions were performed using the following cycle conditions, an initial 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and the final 5 min extension at 72°C. After obtaining the ct-value for each reaction, the relative expression was calculated by 2^-delta Ct method.

Total RNA (5 µg) isolated from rice shoot exposed to different treatments viz., CK, Cd and CdSeL was reverse transcribed by using SuperScriptII (Fermentas, USA). The synthesized cDNA was diluted in DEPC water in the ratio of 1:5 and subjected to quantitative RT-PCR analysis. Each qRT-PCR reaction was performed in a total reaction volume of 20 μl for each set of selected genes by using Fast SYBR Green PCR Master Mix (Agilent Technologies, USA). The qRT-PCR reactions were performed by using the following cycle conditions: an initial 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and the final 5 min extension at 72°C. After obtaining the ct-value for each reaction, the relative expression was calculated by 2^-delta Ct method.

Quantitative expression of some candidate genes involved in alkaloid biosynthesis (Supplementary Table S1) by real time PCR in 20 μl reaction using Fast SYBR Green PCR Master Mix (Agilent Technologies, United States) was performed. Actin was used as an internal control to normalize signal intensity of each transcript in all the reactions. After obtaining the mean Ct value for target and endogenous reference from three biological replicates, the relative expression was calculated by 2^-ΔCt method.