Quantitative PCR Analysis of unc-51 in C. elegans

RNA was isolated from young adult animals. Worms were homogenized, and RNA was extracted with Trizol reagent (Invitrogen) using repeated cycles of vortexing (4 °C) with 500- µm glass beads (Sigma) and freeze-thawing in liquid N₂. RNA was isolated with chloroform (Sigma) and acid phenol:chloroform:Isoamyl alcohol (125:24:1), pH 4.5 (Ambion). Contaminating DNA was removed by subjecting 10 µg of RNA to DNA-free Kit (Ambion). cDNA was generated from 1 µg of RNA with Superscript IV Vilo (Invitrogen). Quantitative PCR was carried out using Power-Up Sybr Green Master Mix in the ABI 7900 cycler (Applied Biosystems). All qPCR amplifications were performed in triplicate using cDNA and no RT negative controls. Quantitation of unc-51 was normalized to tba-1, pmp-3, and Y45F10D.4 reference genes^{75 (link)} using the comparative C(T) method for analysis^{76 (link)}. All primers used for qPCR are listed in Table S5.