Total RNA from adult fly heads or whole flies was extracted with the TRIzol reagent (Applied Biosystems, Grand Island, NY). First strand cDNA pool was made from total RNA (1 μg) with random hexamere oligos SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY) in 20 μl reacting volume. cDNA pool was diluted (1:5) in distilled water. Gene specific assays were used to quantify genes with the SybrGreen method using the PowerSYBR Green Super PCR Mix (ABI Inc., Grand Island, NY) on an ABI7500 machine (Applied Biosystems) using default parameters. Gene specific assays were designed with the PrimeTime qPCR Assay design tool (Integrated DNA Technologies). The housekeeping gene rp49 was used as an RNA loading control as previously described (Lu et al., 2012 (link)). Data were transformed and analyzed according to the ΔΔCt method and are represented as relative fold differences (Lu et al., 2012 (link)). Primer sequences used are: sei-forward: 5′-TTATTCAAAGGCTGTACTCGGG-3′; sei-reverse: 5′-GATGCCATTCGTATAGGTCCAG-3′; ppk29-forward: 5′-CCTCTCAGGTATTCTTCGTTGG-3′; ppk29-reverse: 5′-TCGGTG-GAGATGGTATAGGTC-3′; rp49-forward: 5′-CACCAAGCACTTCATCCG-3′; rp49-reverse: 5′-TCGATCCGTAACCGATGT-3′.
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