We first interrogated reference transcripts for their stable expression by performing RT-qPCR in three technical replicates for each of the five biological replicates on our Strategene MX3005P RT-qPCR machine (Agilent Technologies, CA, USA) using Fast SYBR Green I Master Mix (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions. We performed the PCR in reaction volumes of 10 μl for each replicate consisting of 1 μg cDNA template in three independent replicates with 5 μl of Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) in the presence of 0.4 picomoles of specific primers for the respective candidate reference transcripts. We carried out the reactions in a RT-qPCR thermal cycler (Stratagene MX3005P, Agilent Technologies, CA, USA) according to the manufacturer’s instructions. We involved thermo-cycling conditions that included an initial step of 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 55.0–63.2 °C (Additional file 1: Table S1) for 45 s and 72 °C for 1 min, followed by one cycle of 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s for all the genes. We then assessed stability (non-differential expression) of these reference transcripts using BestKeeper software [43]. From this assessment, we identified gapdh and CLIP-domain serine protease transcripts as less variable [with a standard deviation of crossing point (CP) of 0.56 and 0.65, respectively] among the reference transcript candidates. We thus adopted these two genes as our internal housekeeping transcripts for assessment of expression of the eight randomly selected transcripts. We then separately performed the RT-qPCR for each of these transcripts under similar reaction and thermocycling conditions as had been previously employed in the assessment for stable expression of the reference transcripts above, but with gapdh and CLIP-domain serine protease as internal reference/loading controls.

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