IDT is the largest manufacturer of custom oligo products in the US today. The IDT Customer Care Team frequently receives calls during which representatives are asked to define qPCR terminology. In this article, IDT provides an introduction to some of the most commonly used terms and distinctions encountered in qPCR experiments.
Author- Martin Whitman, Technical support specialist, Integrated DNA Technologies (IDT).
Real-time PCR and quantitative PCR (qPCR)
Real-Time PCR refers to the fact that measurements are made during the amplification as opposed to at the end of PCR. qPCR introduces the idea that the data provides quantification of the target. This is where opinions diverge. Some think that you never really know what you have in a tube (or whether it is available for amplification) and at best relative “quantification” is possible, while others believe true quantification is possible.
That said, the terms are often used interchangeably. Quantitative real-time PCR is often abbreviated as qPCR. Real-time PCR should not be confused with RT-PCR, which refers to reverse transcription PCR, a technique for reverse transcribing RNA into complementary DNA, which is then amplified.
Quantification cycle (Cq) or threshold cycle (Ct)
The cycle at which fluorescence from amplification exceeds the background fluorescence has been referred to as threshold cycle (Ct), crossing point (Cp), and take-off point (TOF) by different instrument manufacturers, but is now standardized by the MIQE guidelines (see Additional Resources for more information) as the quantification cycle. A lower Cq correlates with higher target expression in a sample.
5’ nuclease vs. intercalating dye assays
The two frequently-used variants of qPCR are the 5’ nuclease assay and the intercalating dye assay. 5’ nuclease assays, sometimes referred to as PrimeTime® or TaqMan® assays, exploit the exonuclease activity of Taq DNA polymerase. These assays include two primers, and a probe that is labeled with a fluorescent dye and a quencher. As the Taq polymerase extends from the primers, it encounters and degrades the annealed probe, releasing the dye from the quencher and producing a detectable increase in fluorescence. Multiplex qPCR experiments require use of 5’ nuclease assays where the probes are labeled with different dyes having distinct and separable absorbance spectra.
Intercalating dye assays depend on the ability of dyes such as SYBR® Green, Cyto, EvaGreen®, and LC Green® to fluoresce when intercalated into double-stranded DNA. These assays use only primers and an unbound dye. When new, double-stranded DNA is formed during the reaction, there is a measurable increase in fluorescence as the dye intercalates into the DNA. However, intercalating dyes will interact with any double-stranded product and will fluoresce with non- specific products such as primer-dimers and hairpins. Therefore, it is important to confirm the formation of a single product from intercalating dye assays by analyzing the melt curve of the amplicon or repeating the experiment using a 5’ nuclease assay.
Genomic vs. cDNA assays
The experiments being performed require assays targeted at either genomic (untranscribed) DNA or complementary DNA (cDNA). Typically, researchers measuring gene expression examine the exome, and thus require assays targeting cDNA (created by reverse transcription from mRNA). When designing or ordering an assay, ensure that your assay measures the correct target type.
Tools and reagents for PCR
Integrated DNA Technologies is the world leader in custom oligonucleotide synthesis, using proprietary high fidelity techniques to synthesize oligos of the highest quality. We have applied this expertise in developing our line of PrimeTime qPCR products. For researchers working with human, mouse, or rat genomes, we offer predesigned 5’-nuclease assays with a wide choice of dyes and quenchers as well as predesigned intercalating dye assays. For all other species, our online RealTime PCR Design Tool incorporates a robust algorithm to generate assays targeting the desired gene. ZEN™ and TAO™ Double-Quenched Probes, Available from IDT, will reduce initial background fluorescence and improve performance relative to probes with a single quencher.
IDT also has world-class technical support that is available to answer all types of qPCR questions ranging from experimental design to interpreting qPCR results. Contact us with your questions about qPCR assay design at firstname.lastname@example.org.
Master mixes are mixtures containing most of the reagents required for qPCR. They can be prepared in the lab or purchased from commercial suppliers. Typical components of a master mix include a buffer to maintain pH and salt concentrations, magnesium chloride to stabilize double-stranded interactions and act as a cofactor for Taq polymerase, dNTPs to build the new DNA strands, and Taq polymerase to synthesize the new DNA. IDT recommends Brilliant III Master Mix, available from Agilent Technologies. When performing a 5’ nuclease assay, ensure that you do not use a master mix designed for SYBR® Green or other intercalating dyes, because the fluorescing dyes contained in these master mixes will impair your results.
Several controls are recommended for use in qPCR experiments. The no template control (NTC) monitors contamination and primer-dimer formation that could produce false positive results. For this reaction, simply leave out the cDNA or gDNA template. A no reverse transcriptase control (–RT or no RT) is recommended to monitor genomic DNA contamination when the target sample is cDNA. Another recommended negative control is a no amplification control, where the DNA polymerase is omitted from the reaction to monitor background signal and probe stability.
Two types of positive controls are frequently included in qPCR experiments. The first, an exogenous positive control, is used to check for contaminants in the sample or reaction inhibitors through analysis of dilution series. This is an unrelated sequence, often from the genome of another species, that is spiked into the samples with which you are working.
An endogenous positive control, an assay for a sequence expressed uniformly across all samples (reference genes are often selected for this purpose), is used to correct for quantity and quality differences (normalize) between samples.
A single-nucleotide polymorphism (SNP) is a single DNA base position that varies in nucleotide identity between members of the same species or across paired chromosomes within a single individual. They are the most common type of genetic variation among humans. For such a variation to be considered a legitimate SNP, it must occur with a frequency of at least 1% in the population. While most SNPs occur in noncoding regions and have no effect on the organism carrying them, if present in a coding or regulatory region, SNPs will sometimes impact development and response to disease. They can also serve as biological markers. Researchers often use qPCR assays to detect the presence of SNPs in their samples. Assay design for SNPs is more complex; for more information, contact IDT Technical Support at email@example.com.
Multiplex reactions enable detection of multiple genes in one reaction in a single tube or plate well. For such experiments, each gene must be detected with a probe labelled with a unique dye. Because each dye emits fluorescence at a different wavelength, the key considerations for multiplex design are to ensure that the various primers and probes being used do not interact with each other and to choose dyes that are compatible with your machine.
DECODED 4.2—Special qPCR Issue: Get this compendium of IDT DECODED newsletter articles with qPCR tips, troubleshooting advice, and researcher examples in one convenient volume.
PrimeTime® qPCR Application Guide: Download this useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting chapters. Design Efficient PCR and qPCR Primers and Probes Using Online Tools: Use IDT’s sophisticated, free, online design tools to help you generate your assays. This article will tell you how.
Bustin SA, Benes V, et al. (2009) The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55(4): 611−622; Bustin SA, Beaulieu JF, et al. (2010) MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol.11: 74–78; and Bustin SA, Benes V, et al. (2011) Primer sequence disclosure: A clarification of the MIQE Guidelines. Clin Chem, 57:919−921.