Computational protocol: Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria

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Protocol publication

[…] Eight candidate reference genes (Table ) were evaluated using the validation software geNorm []. These genes had been examined in S. epidermidis and S. aureus in previous studies [,], and analogous sequences were identified in S. pseudintermedius for evaluation in this study. Primers were designed using a combination of GeneRunner software version 3.05 (Hasting Software, Inc.) and the National Centre for Biotechnology Information online primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) using gene sequences for all strains of Staphylococcus pseudintermedius (strains ED99 and HKU10-03) available from Genbank (http://www.ncbi.nlm.nih.gov/genbank/).Primers for icaA were available from a previous study [] and were validated for use in qPCR as described for the potential reference gene primers.Pooled chromosomal DNA from the two strains under investigation (mixture of equal concentrations from each strain) was used to generate dilution series for PCR efficiency calculations and for amplicon melting point assessment. For efficiency determination, a six fold dilution series was prepared in triplicate. Twenty μL reactions containing from 100 ng to 0.1 pg of total DNA were prepared in 96 well plates (Bio-Rad) using the LightCycler 480 SYBR Green I Master qPCR reaction mixture (Roche Applied Science, Indianapolis, IN), following the manufacturer’s instructions. Primer concentration was 0.5 μM for each primer in the final reaction. A Bio-Rad C1000 thermal cycler with a CFX96 Real-Time System and Bio-Rad CFX Manager 2.0 software (Bio-Rad Life Sciences, Mississauga, ON) was used to run the following optimized thermocycling parameters: denaturation at 95°C for 10 minutes followed by 50 cycles of 10 seconds at 95°C, 30 seconds at 62°C, 30 seconds at 72°C, then a melting curve analysis running from 65 to 95°C with steps and measurements every 0.5°C. The thermocycler software calculated quantification cycle (Cq), efficiency and regression coefficients from the recovered data. Amplicon identity and primer specificity was confirmed by sequencing of the PCR products (fluorescent capillary Sanger method, Macrogen, Seoul, Korea), melt curve analysis and gel electrophoresis. qPCR reactions were tested over a range of primer concentrations and annealing temperatures and times to determine optimum reaction conditions. […]

Pipeline specifications

Software tools Primer-BLAST, CFX Manager
Application qPCR
Organisms Staphylococcus pseudintermedius