Rnaqueous isolation kit

Total RNA was isolated using RNAqueous Isolation Kit (Ambion) according to the manufacturer's protocols. TaqMan® Gene Expression Assay primer and probe sets (Applied Biosystems) were used for real-time, quantitative PCR (qPCR) analysis of GAPDH (Hs99999905_m1), PKCζ (Hs00177051_m1), Par6 (Hs00180947_m1) and 18S (Hs99999901_s1). Forward and reverse primer and probe sequences for PKCι were previously described [2 (link)]. qPCR analysis was carried out using 10 ng of cDNA or 2 ng cDNA (18S) on an Applied Biosystems 7900 thermal cycler. Data was evaluated using the SDS 2.3 software package. Gene expression was normalized to GAPDH. All data is expressed as 2^{−(CT(target)−CT(endogenous reference))}.

Strand specific RT-PCR was performed to verify the presence of the replication intermediate negative-strand ZIKV RNA which is a marker of ongoing viral replication. Cellular viral RNA was extracted from the cell lysates harvested at each timepoint using the Ambion RNAqueous Isolation kit. Briefly, 200 uL of binding buffer was added to each well to lyse the cells, which were then removed to a 0.5 mL tube. After adding ethanol, the lysate was gently vortexed and applied to a spin tube to extract the RNA. After washes, the RNA was eluted, aliquoted, and stored at -80° C prior to RT-PCR. cDNA synthesis was conducted using rTth DNA polymerase (Roche Applied Science, Mannheim, Germany) using the method of Lanford, et al. and the primers adapted from Xu.[7 (link), 8 ] For positive-strand detection, the Xu reverse primer was used, and for negative-strand, the forward primer was used. After cDNA synthesis at 55°C and 60°C each for 30 min, 3 uL of cDNA was used in a 50 uL reaction mix containing both primers at 0.2 uM, and Qiagen Taq PCR Master Mix. RT-PCR cycling conditions were 94°C for 1 min, followed by 40 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 10 min. The 113 bp PCR products were observed by agarose gel electrophoresis.