Abi prism 7700 detection system

PCR reactions were performed using an ABI Prism 7700 Detection System (Applied Biosystems, Foster City, CA, USA) with a total volume of 25 ml reaction mixture containing 5 ml of each appropriately diluted RT sample (standard curve points and patient samples), 12.5 μl TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), 900 nM of each primer and 250 nM of the probe. The thermal cycling conditions comprised an initial incubation at 50°C for 2 min, a denaturing step at 95°C for 10 min and 40 cycles at 95°C for 15 s and at 65°C for 1 min. Each experiment included a standard curve with five cDNA concentrations, a control sample (OVCAR3 carcinoma cell-line), 25 patients and no template control. The standard curve was generated using serially diluted solutions of standard cDNA derived from the HTB-77 carcinoma cell line. Real-time PCR assays were conducted in triplicates for each sample. Mean ratios of L1CAM/TBP relative quantitations expressed as arbitrary units were used for calculation.

RNA was extracted by phenol chloroform isolation using TRIzol reagent (Invitrogen) then purified using the MirVANA kit total RNA isolation procedure (Ambion). Following isolation RNA was treated with Turbo DNase (Ambion). cDNA was prepared with SuperScript First-Strand Synthesis System (Invitrogen). qRT-PCR to validate the microarray was performed using SybrGreen master mix (Applied Biosystems). Prognostic datasets were run using TaqMan master mix (Applied Biosystems). Detection was performed using an ABI Prism 7700 detection system (Applied Biosystems). LncRNA expression was normalized to internal control genes TATA-box binding protein (TBP) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Custom-designed primers were used for all SybrGreen reactions. Custom TaqMan primers were used for treRNA and ENST00000413901 TaqMan reactions, with TaqMan TBP primer #4326322E (ThermoFisher) used as the internal control. Custom designed primer sequences are provided in supplemental information (Supplementary Table 1). DeltaCT was determined by subtracting the CT value of the housekeeping gene from the CT value of the lncRNA.