2.5. RNA isolation, DNase treatment, cDNA synthesis, and quantitative PCR

Fat bodies attached to the cuticle dissected from 15 one-week old adult female flies were homogenized in Ribozol RNA Extraction Reagent (Amresco) according to manufacturer's instructions. 5 μg of extracted RNA samples was DNase treated using the DNA-Free Kit (Ambion) and 0.25 μg of DNased RNA samples was reverse transcribed using qScript XLT cDNA Supermix (QuantaBio) according to manufacturer's instructions. cDNA was combined with 200 or 300 nM of forward and reverse primers, 2x Perfecta SYBR Green (QuantaBio) and water to perform quantitative PCR (qPCR). The following genes were amplified: glo (CG6946), rp49 (CG7939), Apoltp (CG15828), Mtp (CG9342), apolpp (CG11064), and bmm (CG5295). Glo and mtp primers were used at 300 nM concentrations while the rest were used at 200 nM. The primers for each gene were:

Glo forward: 5′ GAACCAATCCGACCAGCTAA 3’

Glo reverse: 5′ GCGTCTTTCCGTCGTAGAAC 3’

Rp49 forward: 5′ GACGCTTCAAGGGACAGTATCTG 3’

Rp49 reverse: 5′ AAACGCGGTTCTGCATGAG 3’

Apoltp forward: 5′ GTTCGAGGTGAGTGGTTGGT 3’

Apoltp reverse: 5′ AGCTGCGTCTCATTGGAGAT 3’

Apolpp forward: 5′ ATCGGCTCAACACAAAAACC 3’

Apolpp reverse: 5′ AGGCAAAAGCGATCTCAAAA 3’

Mtp forward: 5′ GTGGGAAGCTTCGTGAAGAG 3’

Mtp reverse: 5′ AAAACGCGATACCATTCGAG 3’

Bmm forward: 5′ ACGTGATCATCTCGGAGTTTG 3’

Bmm reverse: 5′ ATGGTGTTCTCGTCCAGAATG 3’

Quantitative PCR was conducted as follows: 3 min at 95 °C; 40 cycles of: 30 s at 95 °C, 1 min at 60 °C, and 30 s at 72 °C; with a melt curve. Relative quantities were obtained using standard curves and the expression of each gene was normalized to rp49 expression.