Quantitative RT-PCR analysis of gene expression in Caenorhabditis elegans

RNA was isolated with Trizol (TRI REAGENT Sigma), DNAse-treated, and cleaned over a column (RNA Clean and Concentrator ZYMO Research). First-strand cDNA was synthesized in duplicate from each sample (Invitogen SuperScript III). SYBR green was used to perform qRT-PCR (ABI 7900). For each primer set, a standard curve from genomic DNA accompanied the duplicate cDNA samples (Glover-Cutter et al., 2008 (link)). mRNA levels relative to N2 control were determined by normalizing to the number of worms and the geometric mean of three reference genes (cdc-42, pmp-3, and Y45F10D.4; [Hoogewijs et al., 2008 (link)]). At least two biological replicates were examined for each sample. For statistical analysis, one sample t-test, two-tailed, hypothetical mean of 1 was used for comparison using Prism 6.0 software (GraphPad).