Express sybr greener qpcr supermix universal kit

At the same time point, all cell lines were plated into 6-well plates at a density of 1×10⁶ cells/well. Following overnight culture and when 90% of the cells attained confluence, the total RNA was isolated from all cell lines using a PureLink RNA Mini kit (Invitrogen; Thermo Fisher Scientific, Inc.). RNA (1 µg) was reverse transcribed to cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions. Quantitative gene analysis was performed for GAPDH and MIF using an EXPRESS SYBR^® GreenER™ qPCR Supermix Universal kit (Invitrogen; Thermo Fisher Scientific, Inc.) and an icycler iQ5 Real-Time PCR system (Bio-Rad Laboratories, Inc.). The primer sequences used to amplify the cDNA were as follows: GAPDH, forward 5′-CTTAGAGGGACAAGTGGCG-3′ and reverse 5′-ACGCTGAGCCAGTCAGTGTA-3′; MIF, forward 5′-TCGCGAGCTATAGAAGAATCA-3′ and reverse 5′-TGTTCAAGTCTTCGGAGTTTG-3′. Thermal cycling was performed at 95°C for 2.5 min, followed by 45 cycles of amplification at 95°C for 10 sec, 58°C for 10 sec, 72°C for 25 sec and 72 cycles of elongation at 60°C for 5 sec. The data were normalized against the internal GAPDH control in order to obtain ΔCq. Finally, the fold-change of the genes of interest relative to the untreated samples were calculated using the 2^−ΔΔCq method (9 (link)).

Total RNA was isolated from monocytes using a PureLink RNA Mini kit (Invitrogen, Carlsbad, CA, USA), and reverse transcribed to cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories) according to the manufacturers instructions. Quantitative gene analysis was performed for NFATc1, MMP-9 and CTSK using the Express SYBR GreenER qPCR Supermix Universal kit (Invitrogen) and LightCycler 480 Real-time PCR system (Roche). Data were normalized to the internal control GAPDH to obtain ∆Cq. The fold change in genes of interest relative to untreated samples was determined using the 2^−∆∆Cq method (11 (link)). The primer sequences have been previously reported (12 (link),13 (link)).

One microgram of total RNA was reverse transcribed to cDNA in a total volume of 20 μL
using an RT reaction kit (Promega, USA). qRT-PCR was performed using the Express SYBR
GreenER qPCR Supermix Universal Kit (Invitrogen) on a Rotor-gene 6000 system (Qiagen,
Australia). The 25-μL PCR mixture contained 2 μL reverse-transcribed product, 12.5 μL
SYBR Green Supermix, 8.5 μL RNase-free water, and 1 μL each of forward and reverse
primers. The reaction was performed in a 72-well optical plate in triplicate. The
first step of the PCR protocol was 95°C for 10 s, followed by 40 cycles of 95°C for 5
s, and 60°C for 30 s as the second step. A melting-curve analysis was performed to
ensure specificity of the PCR products, and all the PCR products were separated by
electrophoresis on agarose gels to isolate single bands of the expected size. The
expression of TIMs was normalized to β-actin and determined using the comparative
2^-ΔΔCt method (14 (link)). The primers
used were: TIM-1 forward, 5′-GCTTTGCAAAATGCAGTTGA-3′ and reverse, 5′-TGTTGGAATGCCAGATGAAA-3′; TIM-3 forward, 5′-GACTTCACTGCAGCCTTTCC-3′and reverse 5′-GATCCCTGCTCCGATGTAGA-3′; TIM-4 forward, 5′-GGATTTGTGCTCTTCGCATT-3′and reverse, 5′-CCCCCATCCTCAATCTAACA-3′; β-actin forward, 5′-TACAGCTTCACCACCACAGC-3′and reverse 5′-AAGGAAGGCTGGAAAAGAGC-3′; IFN-γ forward, 5′-GCAGAGCCAAATTGTCTCCT-3′and reverse 5′-ATGCTCTTCGACCTCGAAAC-3′.