Quantifying CHIKV RNA by Strand-Specific qPCR

Huh7 cells were infected with CHIKV and treated with inhibitory compounds as described above. At 6 hpi, total RNA was extracted from cells using TRI Reagent Solution (Applied Biosystems) according to the manufacturer’s instructions. Strand-specific qPCR (ssqPCR) was performed according to the protocol described by Plaskon and colleagues [26 (link)]. Briefly, 500 ng of RNA were reverse-transcribed with gene specific primers (S1 Table) using the SCRIPT cDNA Synthesis Kit (Jena Bioscience) according to the manufacturer’s protocol. 100ng of strand-specific cDNA was used as template for the quantitative PCR performed with the qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems) with gene specific primers (S1 Table) amplifying a 94 bp region of the CHIKV nsP1 encoding sequence using the following PCR program: 95°C for 2 mins, 40 x (95°C for 5 sec, 60°C for 30 sec), dissociation curve 60°C-95°C as pre-defined by the Mx3005P thermal cycler (Agilent technologies). In vitro transcribed CHIKV ICRES RNA was reverse transcribed and a cDNA dilution series employed as a standard to quantify copy numbers in the respective samples. All experiments were performed in four independent repeats, each consisting of 2 wells/condition. A one-way ANOVA was employed and Dunnett’s multiple comparisons test was performed comparing each sample to the untreated control sample.