Total RNA was isolated using the GenElute mammalian total RNA kit (Sigma, Madrid, Spain). About 400 ng from each RNA extraction was DNAse treated (Promega, Madison, WI) and reverse transcribed with superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamers (Promega). RNA quantity and quality was estimated by spectrophotometric absorption at 260/280 nm in a NanoDrop ND-1000® spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Expression patterns for B. germanica BgN, BgDl and BgSer were determined by quantitative real-time PCR (qRT-PCR) in sixth-instar nymphs and adults in the first gonadotrophic cycle. Pools of two to six ovary pairs were used in each experiment. PCR primers for use in qRT-PCR expression studies were designed using Primer 3 v. 0.4.0 software [36 (link)]. The actin-5c gene of B. germanica was used as a reference for expression studies, and that of the eukaryotic initiation factor 4A (BgEIF4a) for functional studies. PCRs were performed as previously described [37 (link)]. Primer sequences and the accession numbers the sequences used are shown in the electronic supplementary material, table S1.
Expression patterns for B. germanica BgN, BgDl and BgSer were determined by quantitative real-time PCR (qRT-PCR) in sixth-instar nymphs and adults in the first gonadotrophic cycle. Pools of two to six ovary pairs were used in each experiment. PCR primers for use in qRT-PCR expression studies were designed using P
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