Reverse transcription kit with rnase inhibitor

For the selected genes, specific primers were designed using Primer Blast (NCBI) (Table 2) and purchased from Invitrogen. Hypoxanthine guanine phosphoribosyl transferase (Hprt) was used as a reference gene. Total RNA was treated with the DNA-free kit (Ambion) and cDNA was synthesized from 100 ng of total RNA using High Capacity cDNA, Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems) and random primers according to the manufacturer's instructions. PCR reaction cocktails (25 µl) contained cDNA, Power SYBR green PCR master mix (Applied Biosystems), DEPC-treated water, and custom primers (Invitrogen, Paisley, UK). The PCR reaction was performed in an ABI PRISM 7300 Sequence Detector System (Applied Biosystems). Each reaction was performed in triplicate. The mRNA abundances for each candidate gene were calculated using the following formula: Relative Transcript Abundance = (E target) ΔCt target (control -sample) /(E reference) ΔCt reference (controlsample) (Pfaffl, 2001) , where E is real-time PCR efficiency calculated as Efficiency = 10 (-1/slope) , and control is the mean Ct value of samples of the WT subjects.