Gapdh assay

cDNA was synthesised using a QuantiTec Reverse Transcriptase kit (cat # 205311) according to the manufacturer’s instructions. The plate was incubated for 10 min at 25 °C followed by 1 h at 42 °C and 5 min at 85 °C to inactivate the enzyme on a QuantStudio5 thermocycler. Relative gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and beta (PGC-1β) was determined using the comparative ΔC_T method by calculating the C_T values of the target genes (PGC-1α and PGC-1β) against the C_T values of the reference gene (GAPDH). Both target gene and GAPDH were amplified in same wells, run in triplicate, and respective C_T values were averaged before performing the ΔC_T calculation (ΔC_T = C_TTarget − C_TGAPDH). Gene expression values were converted into log 2 of ΔC_T (2^− ΔC_T).
PGC-1α and PGC-1β expression was measured using a TaqMan® Gene Expression Assay (cat # Hs00173304_m1, Hs00993805_m1; Applied Biosystem) on a QuantStudio 5 qPCR instrument. The total qPCR reaction of 20 µl contained 3 µl cDNA, 10 µl TaqMan® Multiplex Master Mix (cat # 4461882; Applied Biosystem), 1 µl GAPDH Assay (cat # 4485712; Applied Biosystem), 1 µl PGC-1α and PGC-1β Assay and ddH2O. The TaqMan® GAPDH Assay was added to each run as an endogenous control. The thermal profile comprised 95 °C for 20 s, followed by 45 thermal cycles (95 °C for 1 s and 60 °C for 20 s).

RNA was extracted from ARPE-19 cells cultured using an RNA/DNA/Protein Purification Plus Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. The amount and quality of the RNA were determined spectrophotometrically. RNA samples with A260/A280 values of at least 2.0 were used for further analysis. Reverse-transcription of 1 µg of RNA to cDNA was performed using Wonder RT-cDNA Synthesis kit (Euroclone, Milan, Italy). Expression levels of the target genes MerTK (Applied Biosystems, Monza, Italy, assay ID: Hs01031968_m1) and ADAM9 (Applied Biosystems, Monza, Italy, Hs00177638_m1) were measured by qRT-PCR amplification, performed using Luna Universal Probe qPCR Master Mix (New England Biolabs, NEB, Ipswich, MA, USA) in an ABI PRISM 7900 HT Fast Real Time PCR System (Applied Biosystems, Monza, Italy). All reactions were performed in triplicate with the following qRT-PCR run protocol: initial denaturation (95 °C, 1 min), denaturation (95 °C, 15 s), and extension (60 °C, 30 s) repeated 43 times (95 °C, 15 s and 60 °C, 1 min). Gene expression was normalized using β-Actin and GAPDH as control genes (β-Actin assay ID: Hs01060665_g1, GAPDH assay ID: Hs02758991_g1, Applied Biosystems, Monza, Italy). Comparisons of gene expression were done using the 2^−ΔΔCt method [22 (link)].

mRNA expression was determined using quantitative real-time PCR (qPCR). Briefly, total RNA from cells was isolated using TRIzol RNA isolation reagent (Life Technologies, Carlsbad, CA, USA). First-strand cDNA synthesis was performed using the TaqMan Reverse Transcriptase Kit (Applied Biosystems, Branchburg, NJ, USA). Real-time quantitative PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and probes were purchased from the Assays-on-Demand Gene Expression Assay (RRM1 assay ID: Hs00168784_m1; GAPDH assay ID: 4326317E, Applied Biosystems). The comparative threshold cycle method was used to calculate gene expression in the sample, relative to the value in the cells using GAPDH as an internal control for normalization of gene expression among samples. Each assay was performed in triplicate.

Total RNA was extracted from 10⁶ cells using the Trizol® method (Invitrogen) and reverse transcribed using AMV reverse transcriptase (Invitrogen) with random hexanucleotide primers following the manufacturer’s instructions. hTERT mRNA expression levels were quantified by qRT-PCR using an ABI Prism 7900HT Sequence Detection System and TaqMan® Fast Universal Master Mix (Applied Biosystems) [51 (link)]. An hTERT assay (Applied Biosystems), containing hTERT forward (5′-GAGCTGACGTGGAAGATGAGC-3′) and reverse (5′-GGTGAACCTCGTAAGTTTATGCAA-3′) primers and a TaqMan TAMRA™ probe (5′-CACGGTGATCTCTGCCTCTGCTCTCC-3′) giving a 260 bp fragment, and a GAPDH assay (Assay ID: Hs99999905_m1, 124 bp amplicon size, Applied Biosystems), containing GAPDH forward and reverse primers and a TaqMan MGB probe, were used in this experiment. The cycling protocol consisted of 20 s at 95 °C, followed by 50 cycles (95 °C for 1 s and 60 °C for 20 s). Reactions were carried out in triplicate for each sample.