Iscript advanced cdna synthesis kit for rt pcr

Wild–type animals and daf-2 mutants were individually synchronized at 15 °C. Animals were transferred to 20 or 25 °C, after cultivation at 15 °C for 5 days until they passed the L1–L3 larval stage, because daf-2 mutants show a constitutive dauer larva formation phenotype at 25 °C. Messenger RNA was isolated using the RNeasy kit (Qiagen). The isolated messenger RNA (100 ng) was reverse-transcribed to cDNA using the iScript Advanced cDNA Synthesis kit for RT-PCR (Bio–Rad). Quantitative PCR was performed using primers specific to the genes, Sso Fast EvaGreen Supermix (Bio–Rad) and a DNA Engine Peltier Thermal Cycler–CFX96 (Bio–Rad). Triplicate samples of each dilution were used for quantitative PCR. The average expression of each gene was normalized to that of the house–keeping gene lmn-1 as a reference34 (link). The following sets of primers were used for the detection of cgt-1; 5′-GCGATTTTTATGAGACCCGATG-3′ and 5′-CGAACATACAACGCCGAGAA-3′, cpr-1; 5′-AGCTGGATGCAAACCATACC-3′ and 5′-GGAACAGCGTAGGCAGAGAC-3′, dhs-4; 5′-CTTCGATGTTTGACGCCACT-3′ and 5′-CGACCATTGCCTGAGCTTT-3′, dmd-7; 5′-AACGCGAACAGCTAAATGGA-3′ and 5′-GCGTGCCCTTTCAGTACAAC-3′, gpa-7; 5′-TCGACGCAGCAATGAATACC-3′ and 5′-TGCCCTCCAACATCTATCACTC-3′, gpdh-1; 5′-GGGTGACAACGGATTATGAGG-3′ and 5′-CCGACGGAACTTGGGTAGAA-3′, M60.2; 5′-AAGAAGAGCCCAGACCACGA-3′ and 5′-CATCCGAGGCAATGAAGTTGT-3′.