First strand cdna synthesis kit

cDNA was synthesized using the First Strand cDNA synthesis kit (Sigma-Aldrich) according to the manufacturer’s instructions. PCR was performed by using Premix Taq™ (TAKARA) according to the manufacturer’s instructions. Primers used for the Bmi1 gene were 5′-GAGACCAGCAAGTATTGTCC and 5′-TCTTCATCTGCAACCTCTCC. Primers used for the p16INK4a gene were 5′-GAAGGTCCCTCAGACATCCCC and 5′-CCCTGTAGGACCTTCGGTGAC. Primers used for the p14ARF gene were 5′-GTTTTCGTGGTTCACATCCC and 5′-ACCAGCGTGTCCAGGAAG. The GAPDH gene was used as an internal control (primers: 5′-TGCCAAATATGATGACATCAAGAA and 5′-GGAGTGGGTGTCGCTGTTG). PCR was performed using an initial denaturation at 94 °C for 5 min, followed by 18–25 (GAPDH: 18 cycles, Bmi1: 22 cycles, p16INK4a and p14ARF: 25 cycles) cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s. After amplification, an additional elongation step was performed at 72 °C for 10 min. Amplified PCR products were run on 1.5% agarose gels containing 0.5 g/ml ethidium bromide.

Different life stages of B. malayi, viz. adult, mf and L3 were recovered as detailed above. RNA was extracted using the TRIzol reagent (Invitrogen) and quantified with a GeneQuant apparatus (Bio-Rad). After treatment with DNase I to remove genomic DNA contamination, 3 µg of total RNA from each life stage was used for cDNA synthesis using a first-strand cDNA synthesis kit (Sigma-Aldrich, USA). The target gene was amplified using cDNAs applying conditions as mentioned above. For negative controls, PCR was performed with total RNA in absence of reverse transcriptase, in order to rule out any possibility of DNA contamination in the total RNA samples.