Lightcycler 480 2 real time qrt pcr system

The expression of the dengue non-structural NS4A gene in DV2-replicon cells was analyzed using the real-time qRT-PCR. The cells were grown in complete media to reach confluence and then the cells were treated with MPA or ZnS QD-MPA for 24 h. The treated cells were then trypsinized, centrifuged and collected as cell pellets for RNA extraction. Total RNA was extracted from the collected cells using the Trizol reagent method. The concentrations were analyzed using a NanoDrop™ 2000/2000c (Thermo Scientific). The first-strand cDNA was synthesized using a Verso cDNA synthesis kit. The expression of the NS4A gene was measured using Roche SYBR Green RT-PCR kits in a Lightcycler 480 II Real-time qRT-PCR system (Roche). The real-time qRTPCR was performed in triplicate. The mRNA level of GAPDH was measured as an internal control. The oligonucleotides used were
NS4A sense:
5′-AACTGTAGATCTACCATGTCCCTGACCCTGAACCTAATCACAG-3′,
Anti-sense:
5′-TTGACAGCGGCCGCTCATTATCTTTTCTGAGCTTCTCTGGTTG-3′and
GAPDH sense:
5′-AACGGGAAGCTTGTCATCAATGGAAA-3′,
Antisense:
5′-GCATCAGCAGAGGGGGCAGAG-3′.
The qRT-PCR conditions include initial denaturation at 94 °C for 2 min and 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds and elongation at 72 °C for 60 seconds and final extension of 72 °C for 7 min.