Assessing Gene Expression in Caenorhabditis elegans

Approximately 200 synchronized L4 larvae were transferred to fresh NGM plates with or without sulforaphane. After 3 days, adult worms were collected and washed 3× with M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, in 1 L H₂O_bidest) [54 (link)]. The RNeasy Mini Kit (QIAGEN, Manheim, Germany) was used for the extraction of total RNA according to the instructions of the manufacturer. Real-time PCR was performed by using the SYBR Green PCR Master Mix kit (Thermo Scientific, Schwerte, Germany). The PCR primer sequences were designed based on reference to previous studies [62 (link)–64 (link)]. The PCR pairs were synthesized by Eurofins Scientific (Mannheim, Germany), and the sequences are given in Table 3.