Quantstudio 7 flex real time pcr system

RNA was isolated from the mouse RPE following the methodology previously described.²⁶ (link) Total RNA was purified from ARPE-19 cells using the RNeasy Mini Kit (QIAGEN Sciences, Inc., Germantown, MD, USA) following the manufacturer's instructions. Between 100 and 500 ng of total RNA was used for reverse transcription using the Invitrogen SuperScript III First-Strand Synthesis system (Thermo Fisher Scientific, Waltham, MA, USA). The PNPLA2 transcript levels in ARPE-19 cells determined by quantitative RT-PCR were normalized using the QIAGEN QuantiTect SYBR Green PCR Kit in the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The primer sequences used in this study are listed in Table 1. Murine PNPLA2 mRNA levels relative to HPRT transcript levels were measured using the QuantStudio 7 Flex Real-Time PCR System using TaqMan gene expression assays (PNPLA2, Mm00503040_m1; HPRT, Mm00446968_m1; Thermo Fisher Scientific). PNPLA2 relative expression to HPRT was calculated using the comparative ΔΔCT method.²⁷ (link)

Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [31 (link)]. The expression of selected genes was quantified by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems) and are as follows: 5′-ACCTATGACCTGCTTGGTGC-3′ (HIF-1α forward) and 5′-GGCTGTGTCGACTGAGGAAA-3′ (HIF-1α reverse); 5′-TTAAAGCCCGCCTGACAGA-3′ (IL-1β forward) and 5′-GCGAATGACAGAGGGTTTCTTAG-3′ (IL-1β reverse); 5′-TTACAGAGGGAAAACGACACCT-3′ (GPER forward) and 5′-GTGGGTCTTCCTCAGAAGGG-3′ (GPER reverse); 5′-AGTCCCTGAGCATCTACGGT-3′ (COX2 forward) and 5′-CATCATCAGACCAGGCACCA-3′ (COX2 reverse); 5′-AAGCCACCCCACTTCTCTCTAA-3′ (ACTB forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (ACTB reverse). Assays were performed in triplicate and the results were normalized for actin beta (ACTB) expression and then calculated as fold induction of RNA expression.
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific).

Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [29 (link)]. The expression of selected genes was quantified by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Milan, Italy). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems) and are as follows: 5′-CGAGCCCTTTGATGACTTCCT-3′ (c-Fos forward) and 5′-GGAGCGGGCTGTCTCAGA-3′ (c-Fos reverse); 5′-AGCTGTGCATCTACACCGAC-3′ (cyclin D1 forward) and 5′-GAAATCGTGCGGGGTCATTG-3′ (cyclin D1 reverse); 5′-AAGCCACCCCACTTCTCTCTAA-3′ (ACTB forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (ACTB reverse); 5′-CCTTGGAGCCAAATTTAAAACCT-3′ (CXCR4 forward) and 5′-GCTGGACCCTCTGCTCACA-3′ (CXCR4 reverse); 5′-CAGATGCCCATGCCGATTCT-3′ (CXCL12 forward) and 5′-TTCTTCAGCCGGGCTACAAT-3′ (CXCL12 reverse). Assays were performed in triplicate and the results were normalized for actin beta (ACTB) expression and then calculated as fold induction of RNA expression.
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific, Milan, Italy) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Milan, Italy).