These cycles were preceded by a common denaturation step of 2 min at 94°C and followed by a final 10-min extension at 72°C, and were carried out in a Mastercycler ep gradient S thermal cycler (Eppendorf). Amplified products were checked on a 1% agarose gel with a 100-bp marker (Invitrogen) and subsequently CAL 101 purified using the Wizard® SV Gel and PCR
Clean-Up System according to the manufacturer’s instructions (Promega Corporation). Amplified fragments were then cloned in E.coli using the pGEM-T Easy Vector System kit (Promega Corporation), and plasmids from selected clones were purified using PureYield MiniPrep System kit (Promega Corporation) referring to the producer’s manual. Cloned fragments were finally sequenced by Eurofins MWG Operon using primers M13 and sequences were analysed by BLAST alignment [21]. TDF sequences were deposited in the DDBJ database under the accession numbers AB896768 to AB896786. qPCR and data processing qPCR was carried out using the LightCycler SYBR Green system (Roche) as previously described [22]. Briefly, 1 μl of cDNA template was used in each reaction along with 4 μl of SYBR Green PCR master
mix (Roche) and 10 pmol of the appropriate gene-specific primers in a final SBI-0206965 solubility dmso volume of 20 μl. The following cycle profile was used: 10 min at 95°C, 40 repeats Protirelin of 15 s at 95°C, 25 s at 58°C for spxB, ulaE and 16S rDNA genes or 55°C for xfp, 72°C for 20 s (30 s for 16S rDNA) and an additional 5-s incubation step at 81°C for fluorescence acquisition. Oligonucleotide sequence information and detailed primer-specific conditions are given in Table 2. Two technical replicates were done for each combination of cDNA and primer pair. To assess background and residual DNA contamination, a no-template control (NTC) and a no-reverse transcription control (NoRT) were performed for each target. DNA contamination was considered to be LDN-193189 supplier negligible when the
difference in Cq (quantification cycle) between the sample and the respective NoRT was above 5 cycles. Product detection and PCR specificity were checked post-amplification by examining the dissociation curves. PCR amplicons were resolved by 2% agarose gel electrophoresis to verify the expected size. To evaluate repeatability and reproducibility of the qPCR assay, intra- and inter-assay coefficients of variation (CV) were assessed. The intra-assay CV was from 0.7 to 7.6% whereas the inter-assay CV ranged from 8.3 to 18.8%. Amplification efficiency was calculated from the slope of standard curves generated with two-fold serial dilutions of the same cDNA sample, as E = 10(-1/slope). Relative expression of target genes was determined using the ΔΔC T method after Pfaffl correction [23]. 16S rDNA was used as a reference gene.