Qpcrbio probe mix hi rox

RNA was isolated with peqGOLD TriFAST (PeqLab). The reverse transcription was carried out with QuantiTect Reverse Transcription Kit (Qiagen) with indicated primers following manufacturer’s instructions. Relative transcript levels were quantified by quantitative real-time polymerase chain reaction (PCR) in 96-well plates with LightCycler 480 (Roche). The reaction was set by using qPCRBIO Probe Mix Hi-ROX (Nippongenetics) and TaqMan (5′: 6-FAM, 3′: TAMRA) or Universal Probe Library (UPL, Roche) probes. Primers and probes are listed below. Three replicates were used to calculate the mean threshold cycle (Ct) value. The relative enrichments were quantified relative to a housekeeping gene using the reverse transcription primers for cDNA synthesis:
And primers for qPCR are as follows:

Amplifications of TaqMan-based (Thermo Fisher Scientific, Schwerte, Germany) APLNR (Hs00270873_s1), SLC02B1 (Hs01030343_m1), ACE (Hs00174179_m1), MASR (Hs00267157_s1), TNFa (Hs01113624_g1), IL-6 (Hs00985639_m1), ACTB (Hs99999903_m1), and RPL37A (Hs01102345_m1) were performed with qPCRBIO Probe Mix Hi-ROX (Nippon Genetics, Dueren, Germany) in a StepOne plus System (Thermo Fisher Scientific). ACTB and RPL37A (Ribosomal Protein 37a) were employed for normalization. Amplifications of ACE2, AT1R (angiotensin II type I receptor), AT2R (angiotensin II type II receptor), and RPL37A were performed with 5x HOT FIREPol^® EvaGreen^® qPCR Mix Plus (Solis BioDyne, Tartu, Estonia) and sequences in accordance with [23 (link)]. Primer sequences for ACE2 were: S-5′CAT TGG AGC AAG TGT TGG ATC TT, AS-5′GAG CTA ATG CAT GCC ATT CTC A. Data evaluation was performed with DataAssist Software (Thermo Fisher Scientific).
Total RNA for intracellular, monocytic miR-421 was reversely transcribed with TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific) and investigated with RT-PCR by employment of TaqMan (Thermo Fisher Scientific) probes specific for miR-421 (478088_mir) and miR-361-5p (478056_mir), used to normalize the levels of miR-421.