Improm 2 rt kit

Total RNA was extracted using a Qiagen RNeasy kit with DNase treatment according to the manufacturer’s specifications. Reverse transcription (RT) was carried out with the ImProm II RT kit (Promega) using oligodT as primer. Random hexamer was used as primer for NS5 experiments. Real-time PCR analysis was carried out using the SensiMix SYBR & Fluorescein kit (BioLine) and the following primers; mGAPDH 5′-TGCCCAGAACATCATCCCTG-3′ and 5′-ATCCACGACGGACACATTGG-3′, mIL-6 5′-GACTTCACAGAGGATACCACTCC-3′ and 5′-TTCTGCAAGTGCATCATCGGT-3′,mIFNβ 5′-GGAGATGACGGAGAAGATGC-3′ and 5′-CCCAGTGCTGGAGAAATTGT-3′, Kunjin NS5 5′-GAGTCCAAGAAGTCAGAGGGTACA-3′ and 5′-CCACTCTTCATGGTGACAATGTTCC-3′. Reactions were set up in 96-well PCR plates (Genesee). Amplifications were carried out for 50 cycles, followed by a melt curve analysis of resulting products to confirm the specificity of the reactions. To construct standard curves, total RNA was isolated from the cells, and 300- to 600-bp fragments of the gene of interest encompassing the real-time PCR primer binding sites, were amplified by RT-PCR using the appropriate primer sets. PCR fragments were gel purified and quantified, and the copy number was calculated. Serial 10-fold dilutions were prepared for to create standard curves for real-time qPCR. All data are expressed as the ratio of copy numbers of target gene per 10³ or 10⁴copies of GAPDH as indicated.