Qscript cdna supermix kit

Total RNA was extracted from the samples according to the manufacturer’s instructions (Omega Bio-Tek, USA). Samples were washed with PBS and soaked into 350 μL lysis buffer and vortexed. The quality and quality of RNA were measured with NanoDrop Spectrophotometer (NanoDrop Technologies Inc., USA). A total of 1 ug RNA was used for cDNA synthesis using qScript™ cDNA SuperMix kit (Quanta Biosciences, USA). Quantitative real-time PCR was performed as previously described (Pal et al., 2011). In brief, qRT-PCR was performed using CFX96 real time PCR detection system (Bio-Rad Laboratories, Inc, USA). The reaction mix comprises of 10 μL of KAPA SYBR FAST master mix (2×) universal, 0.4 μL of each forward and reverse primers (10 μM), 1 μL of diluted cDNA and PCR grade water to make up a final volume of 20 μL. The following cycling thermal profile was employed: enzyme activation at 95 °C for 3 min, 35 cycles of 95 °C for 3 s and 60 °C for 20 s followed by a dissociation step to analysis the melt curve of the amplified product. ΔΔCt values of triplicates were normalized using endogenous control β-actin, and relative expression was calculated. List of primers used is stated in Supplementary Table S1.

Adult flies were dissected in PBS to collect gonads, male accessory glands, heads, and abdomens. Tissues were immediately transferred to 100 µL of Lysis Buffer with 2% β-mercaptoethanol and stored at −80 °C until required. Total RNA was extracted from these tissues and from different developmental stages (eggs, 2nd, 3rd, and 4th instar larvae, pupae, and adults) using QIAshredder (Qiagen, Valencia, CA, USA) columns to homogenize tissues and a GeneJET RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA). Contaminating genomic DNA was removed using an RNase-free DNase I (Thermo Fisher Scientific) treatment. RNA was quantified and purity was assessed using a Biochrom NanoVue UV-Vis spectrophotometer (Cedarlane Laboratories, Burlington, ON, Canada).
cDNA was synthesized with a qScript cDNA Supermix kit (Quanta Biosciences, Beverly, MA, USA), according to the manufacturer’s protocol. The resulting cDNAs were then PCR amplified using a Lucigen EconoTaq PLUS 2× Master Mix (following manufacturer’s protocol) and specific primers (Table S1, Supplementary Information), and subsequent 1.5% agarose gel electrophoresis. Absence of contaminating genomic DNA was confirmed using no reverse transcriptase in the negative control reactions.

Total RNA was isolated using Trizol reagent (Life Technologies) and treated with the TURBO DNA-free Kit (Life Technologies) to remove residual genomic DNA. Complementary DNA was synthesized using qScript cDNA SuperMix Kit (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer's instructions. Gene specific products were amplified using MyTaq Rad Mix (Bioline USA, Taunton, MA) in a multiple conventional and gradient Veriti Thermal Cycler (Life Technologies) with the following primers: Birc3-f, 5'-GAAACCATTTGGCGTGTTCT-3'; and Birc3-r, 5'-TGGATCGCAATGATGATGTC -3'; Bcl2l1-f, 5'-AATGAACTCTTTCGGGATGGAG-3’; and Bcl2l1-r, 5'- CCAACTTGCAATCCGACTCA-3’; Xiap-f, 5'-CCATGTGTAGTGAAGAAGCCAGAT-3'; and Xiap-r, 5'-TGATCATCAGCCCCTGTGTAGTAG -3'; Actb-f, 5'-CACATCAAGAAGGTGGTG-3'; and Actb-r, 5'-TGTCATACCAGGAAATGA-3'.