Gene Expression Analysis in Synchronized C. elegans

To assess gene expression levels, synchronized young adult worms were grown and frozen in liquid nitrogen. RNA extraction was performed according to a previously published method, with minor modifications [49 (link)]. Briefly, samples were collected and immediately snap frozen in liquid nitrogen. The following steps were performed after the samples were stored at −80 °C. Samples were resuspended in 500 μl of Trizol (catalog no. 10296010, Thermo Fisher). After 3 freeze–thaw cycles, 100 μl of chloroform (Merck, catalog no. C2432) was added. After centrifugation, the upper phase was collected and mixed with the same volume of 70% ethanol. The RNeasy Plus Mini Kit (Qiagen, catalog no. 74134) was used for further total RNA isolation according to the manufacturer’s instructions. Total RNA (500 ng) was used to generate cDNA using the SuperScript IV First-Strand Synthesis System (Invitrogen, catalog no. 18091050, Thermo Fisher). RT-qPCR was performed using SensiFAST SYBR Hi-ROX mix (2x; Bioline, catalog no. BIO-92020) in a LightCycler480 (Roche) with a 96-well white plate (Roche, catalog no. 4729692001). Fold changes in mRNA expression of the target genes were calculated using the ΔΔCt method. The mean expression levels of act-1 and cdc-42 were used as internal controls. The data are presented as mean ± SD (n = 3). The primers that were used in this study are shown in S5 Table.