TaqMan gene primer sets were purchased from Life Technologies (LifeTechnologies, Carlsbad, CA, USA). Reverse transcription and PCR amplification for gene expression assays were performed with TaqMan Transcription and TaqMan Gene Expression Master Mix, respectively, following the manufacturer’s instructions (LifeTechnologies, Carlsbad, CA, USA). RT-qPCR data were normalized, using POL2RA as housekeeping gene.
miRNA RT-qPCR was performed using the miRCURY LNA Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instructions.
Total RNA (20 ng) was polyadenylated and reverse-transcribed at 42°C (60 min), in a reaction volume of 20 μl using a poly-T primer containing a 5′ universal tag. After heat-inactivation at 85°C the resulting cDNA was diluted 80-fold in nuclease free water, and a volume of 8 μl was amplified in 20 μl reaction volume as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 60 s. Normalization was performed with SNORD48 [36 (link)].
Gene and miRNA expression levels were quantified, using a sequence detection system (ABI Prism 7900HT; LifeTechnologies, Carlsbad, CA), and the threshold cycle (Ct) for each sample was determined. ABI SDS 2.4 software (LifeTechnologies, Carlsbad, CA, USA) was used to recover the data, and the relative expression was calculated, using the comparative ΔCt method.
miRNA RT-qPCR was performed using the miRCURY LNA Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instructions.
Total RNA (20 ng) was polyadenylated and reverse-transcribed at 42°C (60 min), in a reaction volume of 20 μl using a poly-T primer containing a 5′ universal tag. After heat-inactivation at 85°C the resulting cDNA was diluted 80-fold in nuclease free water, and a volume of 8 μl was amplified in 20 μl reaction volume as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 60 s. Normalization was performed with SNORD48 [36 (link)].
Gene and miRNA expression levels were quantified, using a sequence detection system (ABI Prism 7900HT; LifeTechnologies, Carlsbad, CA), and the threshold cycle (Ct) for each sample was determined. ABI SDS 2.4 software (LifeTechnologies, Carlsbad, CA, USA) was used to recover the data, and the relative expression was calculated, using the comparative ΔCt method.
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