Prism 7900 ht instrument

Genotyping was performed with the Invader assay (Third Wave Technologies, Madison, WI, USA) and Taq Man Allelic discrimination assay. Reagents were purchased from Applied Biosystems (Foster City, CA, USA). Taq Man probes that can distinguish SNPs after polymerase chain reaction (PCR) were designed and synthesized by Applied Biosystems. One allelic probe was labelled with the fluorescent FAM dye, and the other with the fluorescent VIC dye. PCR was performed with the Taq Man Universal Master Mix with primers at concentrations of 225 nM and Taq Man MGB probes at concentrations of 50 nM. Reactions were performed in 382 well plates in a total volume of 3 μL using 3.0 ng genomic DNA. The plates were then placed in a GeneAmp PCR system 9700 (Applied Biosystems) and heated at 95°C for 10 min, followed by 40 cycles at 92°C for 15 sec and at 60°C for 1 min, with a final incubation at 25°C. The fluorescent intensities of each well in the plates were then read by the Prism 7900HT instrument (Applied Biosystems). Fluorescent data files from each plate were analyzed with the SDS 2.0 allele calling software (Applied Biosystems). Several data points were eliminated to preserve the reliability of the assay system (missing data due to poor signal intensity < 1.1%) [12 (link)].

Total RNA was extracted from cells using Trizol (SIGMA-Aldrich) according to manufacturer's instructions, treated with DNase I (Ambion DNA-free kit, Invitrogen) and converted to cDNA using TaqMan reverse transcription reagents (Applied Biosystems). Quantitative PCR was performed using SYBR-green mastermix (Applied Biosystems and Promega) and run in a Prism 7900 HT instrument (Applied Biosystems). Primers were designed using Primer3Plus, and reactions were done in triplicate. All quantifications were normalised to endogenous HPRT, using the standard ΔΔCt method. Primer sequences are provided in online supplementary table S11.