On column dnase treatment

RNA was extracted from groups of 10 to 50 aphids. Aphids were flash-frozen in liquid nitrogen and ground to a fine powder using a paint shaker (Harbil) and 3 mm steel balls. Following homogenization, RNA was extracted using TRI Reagent (Sigma) and purified with the SV Total RNA Isolation Kit with on-column DNase treatment (Promega). From 1 µg of RNA, cDNA was reverse-transcribed using SMART MMLV reverse transcriptase (Clonetech) and oligo-dT_12–18 as a primer.

Total RNA was extracted from aphid heads using the SV Total RNA Isolation Kit with on-column DNase treatment (Promega), 20 heads per sample. Transcript abundance was analyzed by quantitative real-time reverse transcription qRT-PCR, using ribosomal gene RpL7, ubiquitously expressed and likely a single copy gene, as an internal standard. After extraction and DNAse treatment, 1μg of RNA was reverse transcribed using SMART MMLV reverse transcriptase (Clonetech) and oligo-dT_12–18 as a primer. Gene specific primers were designed using Primer3 (iotools.umassmed.edu/bioapps/primer3_www.cgi). Reactions were performed with 5 μl of 2x Power SYBR Green (Applied Biosystems) and 800 nM primer in the 7900HT instrument (Applied Biosystems) with an initial incubation at 95°C for 10 min. The following cycle was repeated 40 times: 95°C for 15s, 55°C for 15s, and 72°C for 15s and C_T values were quantified and analyzed according to the standard curve method.