The most well-known example of a DNA-binding dye is SYBR Green. The amount of fluorescence emitted upon DNA-binding of the dyes is directly proportional to the amount of dsDNA present. The chemistry of these types of dyes might be simple, but they are extremely effective. Real-time detection of PCR products can be achieved through the use of either: 1) DNA-binding dyes or 2) fluorescent PCR primer- and probe-based chemistries.Īs their name implies, DNA-binding dyes are non-specific fluorescent dyes that intercalate with any double-stranded DNA (dsDNA) molecules. Let’s begin our discussion of probes, shall we? A Little Intro…Īs previously mentioned, the goal of qPCR is to monitor the amplification of a target DNA molecule (either DNA or cDNA) during PCR rather than at the end of the reaction. Whew! I don’t know about you, but I sure feel better having gotten all of that off of my chest. Following reverse transcription of mRNA into cDNA, cDNA is amplified by qPCR and the data can be used to (1) quantify the concentration of the RNA target present in the sample (by using a standard curve), or (2) determine relative quantification for gene expression analysis. RT-qPCR, or reverse-transcriptase quantitative PCR, is, as you probably guessed, a combination of the two methods mentioned above.TIME.) called “real time PCR” and shortened to “RT-PCR”. Thus, it is often (and by often I mean ALL. qPCR refers to quantitative PCR, which allows for the quantification (both full and semi-) of target DNA in real time, as opposed to conventional or end-point PCR, where DNA is quantified at the end of the PCR run.RT-PCR is short for reverse-transcriptase PCR, and is used to detect RNA expression via the use of reverse transcriptase to generate complementary DNA (cDNA).Sooo confusing, amirite? They all refer to specific molecular biology assays, but the names are unfortunately used interchangeably, which can be awfully confusing for just about anyone. Before we go any further, we have to get some things straightened out: RT-PCR versus qPCR versus RT-qPCR.
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