After DNase treatment with Ambion Turbo DNA-free kit (Applied Biosystems, CA, USA), cDNA was synthesised using SuperScript II reverse transcriptase with hexamer random primers (both Invitrogen, CA, USA). Quantification of mRNA transcripts of IL17A, IFNG, IL8 and the reference gene GAPDH was performed using DyNAmo SYBR Green PCR master mix (Finnzymes, Thermo Fisher Scientific, MA, USA) on a Corbett Rotor Gene 3000

system (QIAGEN). Amplification was carried out in triplicate over 40 to 45 cycles of 15 s at 95 °C, 30 s at 61 °C (IFNG, GAPDH) or 62 °C (IL17A, IL8, GAPDH) and 30 s at 72 °C. Included in each assay were commercial human cDNA (Clontech, BD Biosciences, CA, USA) positive controls, no template controls and first-stage RT minus controls. Specificity

Ruxolitinib cell line analysis was performed with high resolution melt curves. Results were analysed by Pfaffl’s relative quantification method ( Pfaffl, 2001), normalising against GAPDH and comparing against a pooled Selleck GSKJ4 negative comparator prepared from a further 14 uninfected donors. Commercial primers were used for IL17A and IFNG (SABiosciences, QIAGEN). IL8 primers were F: 5′-CTCTTGGCAGCCTTCCTGA and R: 5′-AGTTCTTTAGCACTCCTTGGCA. GAPDH primers were as previously described ( Robinson et al., 2008). Data were analysed with Rotor-Gene software (version 6.1, Corbett Research, UK). Statistical analysis was performed using Prism 6.00 (GraphPad, Software CA, USA). Continuous variables were compared using non-parametric Mann–Whitney U-tests. Two-tailed p < 0.05 was considered significant. One of our Benzatropine objectives

was to assess cytokines present at low concentrations and therefore the performance of the three Luminex kits in terms of their sensitivity and assay range. Standard curves provided by each manufacturer were run as recommended but extended to < 1.0 pg/mL to further assess kit sensitivity. As expected all kits performed well within the standard curve ranges recommended by each manufacturer (Table 1), although the Bio-Plex kit was less sensitive for IFNγ in our hands with a lower limit of quantification (LLOQ) of 8.1 pg/mL (vs 1.9 pg/mL lowest recommended standard). The VersaMAP kit had the lowest LLOQ for IFNγ (0.3 pg/mL) although the lowest recommended standard for this kit was 27.2 pg/mL. For IL-17, the Bio-Plex kit was most sensitive with a LLOQ of 1.3 pg/mL. Overall the MILLIPLEX kit performed closest to the specified product characteristics for both analytes. In addition though the upper limits of quantification (ULOQ) were highest with the Bio-Plex kit, the MILLIPLEX kit provided the broadest linear dynamic ranges. Low bead counts for a particular well can reduce confidence in the reported median fluorescence intensity and hence the analyte concentration value interpolated from a standard curve. Manufacturers generally validate their assays with soluble materials such as sera, plasma and cell culture supernatants.