Abi prism 7700 sequence detection system instrument and software

qPCR was performed with ABI PRISM 7700 Sequence Detection System Instrument and software (Applied Biosystems, Foster City, CA, USA), using the manufacturer’s recommended conditions. Total RNA was isolated from transiently transfected cells (TRIzol reagent, Invitrogen, CA), reverse transcribed (Superscript III, Invitrogen, CA), and subjected to quantitative PCR analysis using SYBER green master mix (Invitrogen, CA). The comparative threshold cycle (Ct) method was used to calculate the amplification factor, and the relative amount of targets (Rpl26 and 2810001G20Rik) was normalized to Gapdh levels in parallel reactions. The primer sequences used for qPCR are as follows: Rpl26, 5′-CGA AGC AAG AAC CGC AAA CGG C and 3′-ACC ACC TTG CCA ATC TGC TGG C; 2810001G20Rik, 5′-TGG GAA TGA ACC CTG GCG CTGA and 3′-TTG GGC ACA GCA TCC GTC TTG G; Gapdh, 5′-TTT CCT CGT CCC GTA GAC AAA A and 3′-CGT TGA ATT TGC CGT GAG TGG.

RNA was extracted using Tri Reagent (Sigma), and treated with 10 U DNase (Pharmacia, Milton Keynes, UK). DNase treated RNA (2 μg) was reverse transcribed with M-MLV reverse transcriptase (Promega, Southampton, UK). Multiplex real-time RT-PCR analysis was performed using pre-made TaqMan^® probes (FAM) and 18s rRNA (VIC) specific primers and probes with the ABI PRISM 7700 Sequence Detection System instrument and software (PE Applied Biosystems, Warrington, UK). Expression values were normalized (ΔCt) to 18s rRNA by subtracting the cycle threshold (Ct) value of 18s rRNA from the Ct value of the experimental value.