Principles in qPCR and Data analysis

Quantitative polymerase chain reaction (Q-PCR) is a method by which the amount of the PCR product can be determined, in real-time, and is very useful for investigating gene expression. The qPCR method is sometimes also referred to as real-time PCR or depending on the application, quantitative reverse-transcriptase PCR (both of which are abbreviated to RT-PCR, which can be rather confusing). The main advantage of real-time PCR over PCR is that real-time PCR allows you to determine the initial number of copies of template DNA (the amplification target sequence) with accuracy and high sensitivity over a wide dynamic range. Real-time PCR results can either be qualitative (the presence or absence of a sequence) or quantitative (copy number). Quantitative real-time PCR is thus also known as qPCR analysis. In research laboratories, qPCR assays are widely used for the quantitative measurement of gene copy number (gene dosage) in transformed cell lines or the presence of mutant genes. In combination with reverse-transcription PCR (RT-PCR), qPCR assays can be used to precisely quantitate changes in gene expression, for example, an increase or decrease in expression in response to different environmental conditions or drug treatment, by measuring changes in cellular mRNA levels. This course is on real-time PCR, definition, concept, and types of DNA detection directly using DNA binding dye or indirectly using specific fluorescent dye. What is the suitable method for each experiment? How can we perform the qPCR program? Troubleshooting in qPCR. Data analysis of qPCR output.