title: Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.
abstract: Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression.
authors: Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, 32611-0700, USA.
authors: April M
authors: AM
authors: Lewis
authors: Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, 32611-0700, USA. kcrice@ufl.edu.
authors: Kelly C
authors: KC
authors: Rice
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000235:genetics* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary
keywords: Livak calculation; RNA; Real-time PCR; Relative quantification; SYBR Green; cDNA