cDNA was synthesised using Bioscript™ Reverse Transcriptase and random hexamers (Bioline Pty Ltd, Australia) according to the manufacturer’s protocol. This was the same RNA used for the microarrays. Quantitative PCR (qPCR) was used to validate genes found to be differentially expressed on microarray. Beta Actin (bAct), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), and hypoxanthine guanine phosphoribosyl transferase (Hprt1) were used as normalising genes (Additional file 1 : Table S1). Individual reactions (10 μl) contained 2× Sensifast SYBR Mix (Bioline), forward and reverse primers (10 μM), and 4 μl of cDNA (or 4 μl water for no template controls). The PCR reactions were carried out in a MxPro3005P Real Time PCR System (Stratagene, Agilent Technologies, USA) followed by a dissociation curve. All samples were run in triplicate.
Cycle threshold (Ct) values for each sample were calculated using the MxPRo QPCR software (Stratagene, Agilent Technologies, USA). Triplicate Ct values were averaged and the quantity (Q) of each sample was calculated using the delta-delta Ct method. Q values from the normaliser genes were input into geNorm and the geometric means from these genes were used to generate a normalisation factor (NF) [9 ]. The Q values of the genes of interest were normalised by dividing by the NF value.
Cycle threshold (Ct) values for each sample were calculated using the MxPRo QPCR software (Stratagene, Agilent Technologies, USA). Triplicate Ct values were averaged and the quantity (Q) of each sample was calculated using the delta-delta Ct method. Q values from the normaliser genes were input into geNorm and the geometric means from these genes were used to generate a normalisation factor (NF) [9 ]. The Q values of the genes of interest were normalised by dividing by the NF value.