Kapa probe fast abi prism qpcr kit

RBA-1 cells were seeded on 10-cm culture dishes and treated with 15d-PGJ₂. Total RNA was extracted with TRIzol reagent (Thermo Fisher, Waltham, MA, USA) according to the protocol of the manufacturer. The cDNA obtained from 5 μg total RNA was used to be a template for real-time PCR amplification. Real-time PCR was performed with KAPA PROBE FAST ABI Prism® qPCR kit (KK4705, Kapa Biosystems, Wilmington, MA, USA) and 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the sequences of primers as follows:
Rat NRROS:
Forward primer: 5′-CTCATGCTTCAGAACCTCTCCTG-3′
Reverse primer: 5′-CAGAGTCGTCGAGCCTCATG-3′
Probe: 5′-TGGAGGTCGTGTCCTTGGCAAGAA-3′
Rat IL-6:
Forward primer: 5′-CGAAAGTCAACTCCATCTGCC-3′
Reverse primer: 5′-GGCAACTGGCTGGAAGTCTCT-3′
Probe: 5′-TCAGGAACAGCTATGAAGTTTCTCTCCG-3′
Human NRROS:
Forward primer: 5′-TTTTCACTTCCTGACCGTGG-3′
Reverse primer: 5′-CACCAACTTGCAGACTCCTTG-3′
Probe: 5′-AGGAACAGAAGCGGAACAGCCACA-3′
GAPDH:
Forward primer: 5′-AACTTTGGCATCGTGGAAGG-3′
Reverse primer: 5′-GTGGATGCAGGGATGATGTTC-3′
Probe: 5′-TGACCACAGTCCATGCCATCACTGC-3′
Relative gene expression was determined by the ΔΔ^Ct method, where Ct meant the threshold cycle. All experiments were performed in triplicate.

Total RNA were extracted with TRIzol reagent (Thermo Fisher, Waltham, MA, USA) according to the protocol of the manufacturer. The cDNA obtained from 5 μg total RNA was used to be a template for PCR amplification (Torre-Amione et al., 1996 (link)). Real-time PCR was performed with KAPA PROBE FAST ABI Prism^® qPCR kit (KK4705, Kapa Biosystems, Wilmington, MA, USA) and 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) to analyze the amounts of ICAM-1 and GAPDH mRNA. Fold-changes of gene expression were calculated with the ΔΔCt method and all analysis were performed in triplicate (n = 3).