RNA was prepared from the midgut tissue of the bees, as previously described [25 (link)]. Midgut tissue was manually crushed with a disposable pestle in Trizol Reagent (Invitrogen, San Diego, CA, USA) and RNA was then extracted as per the manufacturer’s instructions. RNA was then DNaseI-treated by RQ1 RNase-Free DNase (Promega, Madison, WI, Canada) and cDNA was synthesized using approximately 1 μg of RNA and the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). For the quantitative PCR (qPCR) reactions to determine the expression levels of the gene of interest, 1 μL of cDNA was used as a template, in conjunction with PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and appropriate primers. Reactions were run in a LightCycler 480 thermal cycler (Basel, Switzerland) or Bio-Rad CFX Opus (Bio-Rad, Hercules, CA, USA) using the PCR conditions stated above. The primer sequences targeting the transcripts of the gene of interest were from [25 (link)]. The difference between the threshold cycle number for β-actin and that of the gene of interest was used to calculate the level of that gene relative to β-actin using the typical 2(−ΔCT) method [23 (link)]. All qPCR data represent the expression values from individual bees (sample sizes found in figure legends) and is displayed as the mean ± SEM.
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