qRT-PCR was performed according to the protocol described previously2 (link). Briefly, cells were first lysed using TRIzol (Invitrogen). Pure RNA was then isolated using the ISOLATE II RNA Mini Kit from Bioline (Alexandria). For tissue samples, snap-frozen tissues were first homogenized using beads (Lysing Matrix D Bulk; MP Biomedicals) in TRIzol. RNA was then isolated as per manufacturer’s instructions using the ISOLATE II RNA Mini Kit from Bioline (Alexandria). 1 μg of RNA was then used to synthesize cDNA, using the Bioline cDNA synthesis kit containing oligo (dT) and random hexamers. All cDNA was then diluted in a 1:10 ratio to perform qRT-PCR.
2.5 μL of diluted cDNA, 0.75 μL of desired primer (2 μM), 3.75 μL of SYBR green (SensiFAST SYBR Lo-ROX kit; Bioline), and 0.5 μL of DNase and RNase free water were mixed and measured on a Real-Time PCR System (Applied Biosystems ViiA 7; Life Technologies Corporation) for 40 cycles. The resulting Ct values were then analysed using the ViiA 7 software (Life Technologies Corporation). The relative expression of target genes was determined by the ΔΔCt method and normalized to housekeeping gene GAPDH or Ywhaz and expressed as a fold difference to the mean of the relevant control samples. See tablesS1 (human) and S2 (mouse) for details on primers used in this study.
2.5 μL of diluted cDNA, 0.75 μL of desired primer (2 μM), 3.75 μL of SYBR green (SensiFAST SYBR Lo-ROX kit; Bioline), and 0.5 μL of DNase and RNase free water were mixed and measured on a Real-Time PCR System (Applied Biosystems ViiA 7; Life Technologies Corporation) for 40 cycles. The resulting Ct values were then analysed using the ViiA 7 software (Life Technologies Corporation). The relative expression of target genes was determined by the ΔΔCt method and normalized to housekeeping gene GAPDH or Ywhaz and expressed as a fold difference to the mean of the relevant control samples. See tables