Mx3000 real time pcr machine

Cells were lysed in Trizol Reagent (Life Technologies) and RNA extracted as indicated by the manufacturers. 1 μg of total RNA was treated with RQ1 DNase (Promega) at 37°C for 30 mins to remove any contaminating cellular DNA, and cDNA generated with the Sensifast cDNA synthesis kit (Bioline) using both oligo d(T) primers and random hexamers. Gene-specific cDNAs were amplified using primers to the WNV_KUN genome and the internal control RPL13A (as previously published [19 (link)]) with ITaq Universal Sybr Green (Bio-Rad) on an MX3000 real-time PCR machine (Agilent). Fold induction of the WNV_KUN genome was calculated by comparing threshold cycle values (CT) to the internal control RPL13A, as previously described [45 (link), 46 (link)].

Total RNA from PHKs was isolated using miRNeasy Mini Kit following the manufacturer’s instructions (Qiagen Inc., Germantown, MD, USA). On-Column DNase I Digestion was performed to prevent genomic DNA contamination. The RT and real-time PCR were performed as previously described (21 (link)). Briefly 1 μg of total RNA was reverse transcribed to 100 μl of cDNA. Samples were run in duplicates, and the PCR were carried out using a Stratagene Mx3000 real-time PCR machine (Agilent Technologies, Inc., Santa Clara, CA, USA). GAPDH was used as the internal reference. The standard –ddCT method was used to measure the relative mRNA expression. 2^-ΔΔCT values were compared using student’s T-test with significance defined as P < 0.05. Primers used in this study were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA). Primers used are provided in Supplementary Table 1.