Perfectin transfection reagent

Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics (Laval, Quebec, Canada); ProLong Gold anti-fade reagent with 4,6-diamidino-2-phenylindole (DAPI), SlowFade Gold reagent mounting media, cell culture media DMEM, RPMI 1640, and fetal bovine serum (FBS) from Invitrogen (Carlsbad, CA) were purchased from the indicated suppliers.
Lipofectamine 2000 and Lipofectamine RNAiMAX were purchased from Invitrogen (Carlsbad, CA); Per-Fectin transfection reagent was from Genlantis (San Diego, CA).
miRIDIAN microRNA mimics including human hsa-miR-500a-5p, hsa-miR-34c-3p, hsa-miR-93-3p, hsa-miR-381-3p; miRIDIAN microRNA Mimic Negative Control #1 and miRIDIAN microRNA mimic mouse mmu-miR-344-3p; miRIDIAN microRNA inhibitors including human hsa-miR-500a-5p-Hairpin Inhibitor, hsa-miR-34c-3p-Hairpin Inhibitor, hsa-miR-93-3p-Hairpin Inhibitor and hsa-miR-381-3p-Hairpin Inhibitor were purchased from GE Healthcare Dharmacon Inc. (Lafayette, CO).
MGC human PEX2 (Clone ID: 3347824), PEX7 (Clone ID: 5176358), PEX11B (Clone ID: 3866690), and PEX13 (Clone ID: 6285875) sequence-verified full-length cDNA clones were purchased from GE Healthcare Dharmacon Inc. (Lafayette, CO).
Reagents for purification and quantitation of miRNAs including MiRNeasy Mini kit, miScript PCR Starter kit, miScript II RT kit, and miScript SYBR Green PCR kit were purchased from Qiagen (Toronto, ON).

RCC cells were grown after thawing in normal medium (RPMI1640, 2 mM L-Glutamin, 100 μg/ml Streptomycin, 10% FCS, Biochrom, Berlin, Germany) for 7 days. After passaging cells were treated on day 2–4 with 0,125 μM 5-aza-2`desoxycytidine (Sigma, St. Louis, USA) or a mock solution (control cells) and cultured in normal medium on days 5–7 until harvesting. Isolation of total RNA, reverse transcription and relative quantitation of mRNA levels of internal controls as well CRHBP mRNA were carried out as described before [7 (link)].
For ectopic expression of CRHBP 3μg of human cDNA expression vector (NM_001882, Origene Technologies, Rockville, MD, USA) and PerFectin transfection reagent (T303007, Genlantis, San Diego, CA, USA) were applied according to the manufacturer´s instructions in a transfection volume of 1 ml for 6*10⁵ cells
CHO cells were used as control for transfection and expression efficiency. For SDS-PAGE and Western blot analysis each 0,5 x 10⁶ transfected cells were lysed in 100 μl 2x sample buffer (125 mM Tris-HCl pH 6,8, 20% Glycerol, 4% SDS, 5% Mercaptoethanol, 0,025% Bromphenolblue). Immunoprobing was carried out by use of the anti-Flag-POD antibody (A8592, Sigma-Aldrich, St. Louis, MO, USA).