Real-time quantitative PCR has been applied for many different biological applications including identifying genomic DNA copy number, single nucleotide polymorphism (SNPs), DNA methylation status, viral load and many others. One of the more popular applications of real-time PCR is to quantify levels of RNA (Wong et al., 2005). Using this technique, RNA is first converted to cDNA and then amplified by the PCR. The products of the PCR are detected in real-time using dedicated instrumentation.
Quantifying gene expression using PCR is one of the more intimidating techniques of modern molecular biology because of the perception that the RNA will be degraded and/or inaccurately measured. The advent of realtime quantitative PCR has greatly simplified gene expression analysis. The purpose of this protocol is to describe each of the steps that are involved in measuring gene expression using real-time PCR. These include sample preparation, isolation of RNA, reverse transcription, primer design, plate set-up and data analysis. Variations of the protocol are included for both cell lines and tissues. An emphasis has been placed on various tips and tricks to increase throughput and reduce error. Implementation of a well-organized and methodical protocol will produce accurate, precise and reproducible data no matter what the tissue or gene of interest.
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