Rotor gene q cycler

Total RNA was isolated from wild type or transfected HEK293T cells using Quick-Zol reagent (Kalium Technologies, Bernal, Argentina) following the manufacturer’s instructions. For the first-strand cDNA synthesis, 1 μg of total RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems^TM, Beverly, MA, United States) with random primers. Quantitative real-time PCR (qPCR) was performed in triplicate on the Rotor Gene Q cycler (Qiagen, Hilden, Germany) using the resulting cDNA, the HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) for product detection, and the following primers: human MasR (NM_002377.2) forward, 5′-ATTCCTCATCTTCGCTATGCC -3′ and reverse 5′-GCAGGGAAATGTGGTGTAGG-3′; human AT1R (M93394.1) forward, 5′-CCCAAAATTCAACCCTTCCG-3′ and reverse, 5′-CAGAAAAGGAAACAGGAAACCC-3′; human AT2R (NM_000686.4) forward, 5′-CCCTGAACATGTTTGCAAGC-3′ and reverse 5′-AGGGGTAGATGACAGATTGG-3′; and human β-Actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′ and reverse 5′-AGCACTGTGTTGGCGTACAG-3′. The cDNA was amplified by 45 cycles of denaturing (15 s at 95°C), annealing (30 s at 60°C), and extension (30 s at 72°C) steps. The specificity of each primer set was monitored by analyzing the dissociation curve, and the relative MasR, AT1R or AT2R mRNA quantification was performed using the comparative ΔΔCt method using β-Actin as the housekeeping gene.

The expressions of pluripotent markers, cluster of differentiation (CD) markers, and lineage-specific genes were analyzed. An easy-spin Total RNA Extraction Kit (Intron, Seongnam, Korea) was used for total RNA extraction. Nucleotide quantification was performed with a nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA was synthesized with 2 μg purified RNA and reverse transcription with a HisenScript RT PreMix kit (Intron, Seongnam, Korea); synthesis was performed over 50 min at 42 °C with 10 μM OligodT primers. A Rotor-Gene Q cycler (Qiagen, Hilden, Germany) was used for RT qPCR and RealMODTM Green AP 5× qPCR mix (Intron, Seongnam, Korea) containing 200 nM primers (Table 1). Amplification was performed by denaturation at 95 °C for 60 s and subsequently 50 cycles of 95 for 10 min, 60 °C for 6 s, and 72 °C for 4 s. Expression was normalized to mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR products were analyzed by electrophoresis in 1% agarose gels and ethidium bromide staining. Amplified products were visualized under UV light. All experiments were conducted in 4 independent replicates.

Target gene expression levels were quantified from cDNA using the 5× HOT FIREPol^® EvaGreen^® qPCR Mix Plus (Medibena) on a Rotor-Gene Q cycler (Qiagen) with gene-specific primers: GAPDH (human): 5′-CGACCACTTTGTCAAGCTCA-3′ and 5′-TGTGAGGAGGGGAGATTCAG-3′, Actb (mouse): 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′, NSUN5 (human): 5′-CTACCATGAGGTCCACTACAT-3′ and 5′-CTGGCAGAGGGAGCA-3′, Nsun5 (mouse): 5′-TTGCAAGAGAGCTCCAGACC-3′ and 5′-AGGCAGCAAGGGATCCAAAA-3′.