For real-time SYBR-Green based qPCR total RNA was extracted using NucleoSpin RNA Plus kit (Macherey-Nagel). Total RNA was reverse-transcribed into cDNA by M-MLV Reverse Transcriptase (Promega) in RT reaction containing Random hexamers (Promega), Oligo (dT) 18 Primer (Thermo Scientific), the mix of all 4 dNTPs and Riboblock RNAse inhibitor (Thermo Scientific). The cDNA amount was determined as the synthesized cDNA in a 20 μl RT-reaction containing 1 μg total RNA.
Gene expression was assessed using SYBR-Green based qRT-PCR. The reactions for the qPCR were prepared with a Corbett CAS-1200 liquid handling system and the qPCR was performed using Corbett Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) with a thermal cycle of 95°C for 15 min, followed by 40 cycles of 95°C 25 s, 60°C 25 s, 72°C 25 s, followed by a melting step. Relative quantification of gene expression was performed following the ΔΔCt method with housekeeping gene Cyclophilin G as an endogenous control. All qPCR primers are listed inTable S12 .
Gene expression was assessed using SYBR-Green based qRT-PCR. The reactions for the qPCR were prepared with a Corbett CAS-1200 liquid handling system and the qPCR was performed using Corbett Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) with a thermal cycle of 95°C for 15 min, followed by 40 cycles of 95°C 25 s, 60°C 25 s, 72°C 25 s, followed by a melting step. Relative quantification of gene expression was performed following the ΔΔCt method with housekeeping gene Cyclophilin G as an endogenous control. All qPCR primers are listed in
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