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2.4. RNA extraction and reverse transcription (RT)
The mRNA extraction and the cDNA synthesis were performed using the Cells-to-cDNA kit (Ambion) according to manufacturer′s instructions. The Verteporfin were lysed in 100 μL of ice-cold cell lysis solution, followed by heat treatment at 75 °C for 15 min. Traces of genomic DNA were eliminated with DNase treatment at 37 °C for 15 min, followed by 5 min of heat inactivation at 75 °C. Two cDNA synthesis reactions from each replicate well were performed. Briefly, 2 μL of dNTPs and 1 μL of random hexamers were mixed with 5 μL of RNA, followed by incubation at 75 °C for 3 min. Two microliters of master mix composed of 1 μL of 10× reverse transcription buffer, 0.5 μL of M-MLV reverse transcriptase and 0.5 μL of RNase inhibitor were added and incubated at 42 °C for 60 min and 95 °C for 10 min.
2.5. Quantitative real-time PCR (qPCR)
For qPCR, reactions were performed in triplicates in a final volume of 5 μL; using 2 μL of cDNA diluted 20-fold with dH2O, 2.5 μL of Fast 2× TaqMan master mix and 0.5 μL of gene-specific TaqMan assays (Applied Biosystems) (see Supplementary material File 1). Reactions were run using the StepOne Plus Real-Time PCR System (Applied Biosystems) using the manufacturer’s conditions. All reactions had a PCR efficiency value (E ) of 2 ± 0.03 approaching 100%. To normalize data for geNorm, C t values were transformed to relative gene expression values using the equation EΔCt(Ctmin-Ctsample) [21].