This facility was established by a grant from Health Resources and Services Administration (HRSA) Charles E. Schmidt College of Science Division of Research & Graduate Studies

Introduction

Microarray expression profiling examines changes in gene expression. Real-time Reverse Transcriptase-PCR may be used to validate differentially expressed genes and quantify fold changes relating to microarray data. The Lightcycler™ (LC) facilitates rapid cycling using hot air via a thermal chamber, and measures fluorescence in specially designed glass capillaries that contain the PCR reaction components. PCR amplification profiles are composed of 3 segments: an early background phase (little product accumulation), an exponential phase ( or log linear phase, rapid product accumulation) and a plateau phase (no further product is amplified). Real-time PCR works on the principle of measuring fluorescence signals at the end of the extension phase of each PCR cycle that are generated following the binding of SYBR Green I, a dsDNA-binding dye, to PCR products. As the number of product increases, so too does the fluorescence signal, resulting in a characteristic sigmoidal-shaped curve. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and by empirically determining annealing and signal acquisition temperatures for each gene-specific primer pair. Relative expression levels are quantified by constructing a standard curve using serial dilutions of a cDNA template to monitor the expression of a highly expressed gene.