Five single larvae per tank and time point were weighed, photographed
and each homogenized in 1 ml Tri-Reagent (Sigma,
Germany). Photographs were used to determine length and width
of the larvae. After obtaining the aqueous phase by incubation with
chloroform, RNA was extracted from this phase using the RNeasy
Mini kit (Qiagen, Germany) following the manufacturer's instructions.
The RNA concentration was determined by Nanodrop
ND-1000 (Peqlab, Germany) and normalized to a common
concentration with RNase free water before proceeding with cDNA
synthesis. 240 ng RNA were transcribed with the Quantitec kit
(Qiagen, Germany) according to the manufacturer's instructions
including a genomic DNA wipe-out step. Controls for gDNA were
also included and cDNA was stored at 20 C until further use.