RNA Extraction, cDNA Synthesis and qPCR

Total RNA was extracted for each experimental condition from 50ul of Drosophila embryos (collected 14–16hrs after deposition) using RNeasy Mini Kit (Qiagen) as per the manufacturer’s protocol. Total RNA concentration was measured using NanoDrop ND-2000 Spectrophotometer and the purity of the samples was determined by the OD ratios, A₂₆₀/A₂₈₀. One µg of total RNA was reverse transcribed in a 20 µl reaction volume using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s protocol. Gene specific primer sequences were obtained from Fly Primer Bank

(en forward primer, 5′-TCCGTGATCGGTGACATGAGT-3′;

en reverse primer, 5′-CGCCGACGTATCATCCACATC-3′;

wg forward primer, 5′-GACCCAGCGATCCACTCTAC-3′;

wg reverse primer, 5′-CGGCGATTTCTGAACTGGTGT-3′;

HA forward primer, 5′-GTTCCTGACTATGCGGGCTA-3′;

HA reverse primer, 5′-AGCGTAATCTGGAACGTCAT-3′;

RpL32 forward primer, 5′-CCCAAGGGTATCGACAACAGA-3′;

RpL32 reverse primer, 5′-CGATCTCGCCGCAGTAAAC-3′)⁷⁸.

Quantitation of mRNA was performed using SYBR® Green Assay (Thermo Fisher Scientific) on the PikoReal™ Real-Time PCR System (Thermo Fisher Scientific) and a PCR product dissociation curve was generated to ensure specificity of amplification. RpL32 was used as an endogenous control and relative quantitation was performed using relative quantification (2^−ΔΔCT). Results were generated from 3 technical replicates for each mRNA. The average relative expression ± standard deviation (SD) was determined and two sample t-test was carried out to determine statistical significance.