Lightcycler 480 probe master

Primers and probes (Table 1) were purchased from Eurogentec. Renal DNA was amplified using the LightCycler FastStart DNA Master SYBR Green I and targeted the lfb1 gene [43 (link)] on a LightCycler 2.0 (Roche Applied Science). Alternatively, bacterial load was assessed using the LightCycler 480 Probe Master targeting lipL32 gene on a LightCycler 480 II instrument (software v.1.5.0; Roche Applied Science) [44 (link)]. Genomic DNA from corresponding leptospires was used as a positive control. Quantitative PCR for transcripts of the cytokines IL-1β, IL-10, TNF-α, TGF-β, iNOS, the chemokines IP-10/CXCL10 and MIP-1α/CCL3, and for reference genes glyceraldehyde-3-phospho-deshydrogenase (GAPDH) and β–actin were conducted from cDNA on a LightCycler 480 II using the LightCycler 480 SYBR Green I Master. Each qPCR was carried out with 2 μL of cDNA or gDNA in 20 μL final volume following gene-specific amplification programs (detailed in Table 1) and the specificity of SYBR Green I-based qPCR assays was verified by the melting temperature (Tm) of the amplicon as calculated by the instrument software (see Table 1). Results were validated only when threshold cycle (Ct) values were under the limit value of 40 cycles and with an acceptable reproducibility between qPCR replicates (less than 5% of variation).

The mRNA expression was examined by qRT-PCR as described previously [10 (link)]. qRT-PCR was performed using a LightCycler 480 Probe Master (Roche Diagnostics, Tokyo, Japan) and Universal ProbeLibrary (Roche Diagnostics) probes or a LightCycler 480 SYBR Green I Master (Roche Diagnostics) on a LightCycler 480 System II (Roche Diagnostics). The primers and probes used are listed in Supplementary Table S1. Amplification was performed in a two-step procedure and mRNA levels were measured quantitatively using the Δ/Δ threshold cycle method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1 (PGK1) were used as controls. Triplicate wells were analyzed for each assay.

Cs shRNA and Con shRNA cells were homogenized in 1 ml of ice cold TRIZOL Reagent (Invitrogen Ltd, Paisley, UK) and RNA extracted using chloroform and isopropanol as described previously [6 (link)]. 2 μg of RNA was then used for cDNA synthesis in 20 μl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 0.5 mM dNTP Mix (0.5 mM each dATP, dGTP, dCTP and dTTP), 5 mM DTT, 150 ng of random primers, and 200 units of SuperScript™ III Reverse Transcriptase. Real time PCR was performed using Roche Lightcycler 480 II (Roche Diagnostics, Sussex, UK) and Multiplex Taqman assays for Cs as a target gene and β-actin as a reference gene in each sample. Three μL of cDNA was added to 10 μL of LightCycler 480 Probe Master (Roche), 0.2 μL of TaqMan probe (Probe no. 100, Universal Probe Library), 0.2 μL of forward and reverse primers (20 μM) each, and 1 μL of mouse β-actin probe dye VIC-MGB (Applied Biosystems, 4326317E). The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-Genosys (forward primer: 5′-GGAAGGCTAAGAACCCTTGG-3′ and reverse primer: 5′-TCATCTCCGTCATGCCATAGT-3′) and the corresponding UPL probe (UPL probe #100) was used. The results were analysed using LightCycler® 480 software 1.5 and Cs was normalized to β-actin and presented as a ratio (ratio = (1 + E_Cs)^−Ct(Cs)/(1 + E_β-actin)^{−Ct(β-actin)}).