Ageing, immunity and stress tolerance are inherent characteristics of all organisms. These traits in Caenorhabditis elegans have been shown to be regulated at least in part by the daf-16 gene. Through our research we aimed to establish a correlation between the expression of daf-16 and observed phenotypes (lifespan, immunity and stress tolerance) among four closely related Caenorhabditis species (C. elegans, C. briggsae, C. remanei and C. brenerri). For such studies we required optimal concentration of high quality extracted RNA with minimal genomic DNA contamination. The added complication of using entire organisms for RNA extraction also meant that a very effective method be used to produce homogenized samples for RNA Isolation.
Employing this methodology we were able to successfully isolate required amounts and quality of RNA from four species of the Ceanorhabditis genus at various stages of development. On average a yield of 1.5 – 2 µg/µl of RNA was obtained per 9cm petri plate. This RNA was then used for RT-PCR to quantify gene expression studies as shown in Figure 1.
Figure 1: Expression levels of daf-16 (normalized to the reference gene gpd-3) among mixed populations – nematodes at various stages of development adapted from Amrit et al, 2010 .