RNA-extraction and RT-PCR

Rh30 (human aRMS) cell line and murine aRMS primary tumor cell cultures (U23674) were treated with 0.1, 0.2, 0.4, 0.8, 1, and 2 μM ENT for 24 hours. DMSO treatment was used as a control. After treatment with ENT, total RNA was extracted and complementary DNA (cDNA) was synthesized as previously described (13). Expression of PAX3:FOXO1 was determined by real-time PCR (RT-PCR) using custom Taqman primers and probe (catalog nos. 4304970 and 4316034) on a StepOnePlus RT-PCR machine (Applied Biosystems). Rh30 and Rh41 cell lines were treated with 1 μM ENT, 45 nM PAN, and 1 μM SAHA for 24 hours. DMSO-treated cells were used as control. Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was prepared from RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems) with a ribonuclease inhibitor. qPCR was performed using TaqMan Universal Master Mix, no AmpErase Uracil-N glycosylase on the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). Primers used were Gapdh-Hs02758991_g1 and PAX3:FOXO1-Hs03024825_ft. Gene expression was quantified using the 2^−ΔCt method.