Total RNA was extracted from leaf samples of C. cajan by Trizol method and quantified by Thermo Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). One mg of RNA was used for the synthesis of cDNA using the First-Strand Synthesis kit. cDNA was stored at −20 °C for further analysis. qRT-PCR was performed for expression analysis of transcripts level using a qRT-PCR detection system (CFX96 Touch™ Real-Time PCR Detection System, Bio-Rad laboratories, Hercules, CA, USA) with iTaq Universal SYBR Green SuperMix. The “Oligo Calculator”, an online tool (http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html (accessed on 14 August 2021)), was used to design gene-specific primers, which were further verified by the NCBI-primer BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 14 August 2021)). The expression analysis was triplicated for each of the genes and the GAPDH gene was considered as the housekeeping gene [67 (link)].
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