Validation of splicing changes using semi-quantitative RT-PCR

For preparation of L4-staged samples, worms were synchronized using the bleaching method and an additional sorting step was done on a Union Biometrica COPAS worm sorter using an empirically determined window of TOF and EXT values that captures L4 worms. RNA was isolated using TRIzol reagent (Invitrogen) using standard RNA extraction protocols. After digestion with DNase I (Sigma), random nonamers (Sigma) were used to synthesize total RNA into cDNA using SuperScript III reverse transcriptase (Invitrogen). PCR amplification cycles used ranged from 27–35 cycles, and PSI values were estimated by densitometric analysis using ImageJ [101].