Revertaid h minus rt revert transcriptase

For real-time PCR experiments, total RNA was isolated from S2 cells with TRIzol reagent (Invitrogen). Genomic DNA was removed by treatment with DNase I (Fermentas, 1 U per 10 μg) followed by purification with a QIAGEN RNeasy kit. RNA was reverse transcribed into cDNA with a RevertAid H Minus RT Revert Transcriptase (Fermentas) following the manufacturer’s instructions.
The resulting complementary DNA (cDNA) was analyzed by quantitative PCR (Bio-Rad CFX 96 Cycler) using SYBR Green. Relative steady-state mRNA levels were determined from the threshold cycle for amplification by the ΔΔCT method. For each experiment, duplicate or triplicate reactions were performed and averaged, using two independent RNA samples. The expression level of each gene was determined using ras64B, RpL32, eh, and β-Tubulin56D as an internal control. Primer sequences used in real-time PCR analysis are listed in S4 Table.

For RT-qPCR experiments, total RNA from the heads and ovaries of females was isolated with TRIzol reagent (Invitrogen). Genomic DNA was removed by treatment with DNase I (Fermentas, 1 U per 10 μg) followed by purification with a QIAGEN RNeasy kit. RNA was reverse transcribed into cDNA with a RevertAid H Minus RT Revert Transcriptase (Fermentas) following the manufacturer’s instructions. The resulting cDNA was analyzed by quantitative PCR (Bio-Rad CFX 96 Cycler) using SYBR Green. Relative steady-state mRNA levels were determined from the threshold cycle for amplification using the ΔΔCT method. Each experiment was performed in two to three independent biological replicates, and the results were averaged. The expression level of each gene was determined using ras64B as an internal control. Primer sequences used in real-time PCR analysis are listed in Suppl. Table 2.