Real-Time PCR for Gene Expression Analysis

After discovery of polymerase chain reaction (PCR) by Dr. Kary Mullis in 1983, several different types of PCR have been invented and continually improved upon over the years. One of them called "Real-time PCR" or “fluorescence based PCR” allows us to quantitate nucleic acids obtained from cells or tissues, to compare the variable states of infection, to detect chromosomal translocations, to genotype single nucleotide polymorphisms, to determine gene expression level of samples and so on. For the detection and quantification of nucleic acids, Real-time PCR has become the most accurate and sensitive method. Quantitative measurement of specific gene expression using quantitative PCR (qPCR) is necessary for understanding basic cellular mechanisms and detecting of alteration in gene expression levels in response to specific biological stimuli (e.g., growth factor or pharmacological agent) (Bustin, 2000; Bustin, 2002). Quantification of nucleic acids has been significantly simplified by the development of the Real-time PCR technique (Bustin, 2002; Huggett et al., 2005). It is mostly used for two reasons: either as a primary investigative tool to determine gene expression or as a secondary tool to validate the results of DNA microarrays (Valasek & Repa, 2005).