Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Suppose you have two genomic DNA samples. You want to know if they contain evidence of a given pathogen. What do you do? The answer, of course, is PCR. The Nobel Prize-winning DNA amplification method is the go-to method for applications like this.
But what if a qualitative answer isn’t enough? Now you want to know not only whether the pathogen’s genome is present, but also at what abundance. Again, the answer is PCR. But not PCR of the same type.
To simply determine whether or not a specific sequence is present in a sample (as in the first question), you generally use traditional PCR, which is an endpoint assay. Mix template, primers, and reagents in a tube, amplify for 30 or 35 cycles, and run the products out on a gel. Barring contamination and other technical problems, if you see a band, the sequence is there.
The problem, explains Tania Nolan, Global Manager for Applications and Technical Support at Sigma Custom Products, part of Sigma-Aldrich, is that the intensity of those bands is neither an absolute nor even a relative indication of starting concentration.
“If you have two samples, one containing 1,000 target copies of your gene of interest and another containing 10,000 copies, then it’s highly unlikely you would then be able to run that product on a gel and say definitively that I have 10-times more copies in one sample than the other,” Nolan says. “You would be able to say, ‘I’m sure I’ve got my target in both,’ but you wouldn’t know relative amounts.”
Which leads us to quantitative PCR (qPCR, also called real-time PCR), the tool to address questions of abundance.
qPCR is a fluorescence-based, quantitative assay. As the reaction runs, its fluorescence intensity changes. A qPCR instrument monitors that fluorescence as it cycles, recording precisely when the detected fluorescence crosses a threshold value, sometimes called the threshold cycle (Ct) or quantification cycle (Cq). It is this threshold value that makes the reaction quantifiable -- theoretically, as PCR doubles a target sequence every cycle, two samples that differed by one Ct unit would have a two-fold difference in starting copy number.
Researchers use qPCR for such applications as quantifying RNA abundance, an application called reverse transcriptase (RT)-qPCR, validating microarray or sequencing data, quantifying pathogen loads, and genotyping. Fortunately, there’s no shortage of tools to perform these analyses. If you have the need, you should have no problem finding the reagents to address them.
Dye- vs. probe-based qPCR
qPCR reactions basically come in two flavors. The simplest, and least expensive, is the dye-based approach. Generally accomplished using a DNA dye called SYBR® Green (or alternatives, such as Promega’s BRYT® Green) the dye-based qPCR measures total double-stranded DNA during the amplification process, regardless of sequence. (Some formulations also include an internal fluorescent reference standard, such as ROX, to correct for background.)
Dye-based reactions
The advantages of dye-based qPCR reactions are largely financial. Dyes are relatively inexpensive, and will work with any sequence. But they also cannot be easily multiplexed. Also, dye-based reactions cannot distinguish amplification of the intended target from off-target effects such as primer-dimers, except via melt curve analysis.
According to Surekha Karudapuram, Associate Marketing Director in the Diagnostics and Genomics Group at Agilent Technologies, melt curves enable users to determine if a sample contains one or multiple fluorescent species. “That tells you how specific the amplification is,” Karudapuram says. She adds that melt curves also theoretically enable users to multiplex dye-based reactions, and even to roughly quantify those peaks.
Dye-based master mix reagents are available from Agilent Technologies (Brilliant III Ultra-Fast SYBR® Green QPCR & QRT-PCR master mixes), Bio-Rad Laboratories (e.g. SsoAdvanced™ SYBR® Green Supermix), Life Technologies (SYBR® Select Master Mix), Promega (e.g., GoTaq® qPCR Master Mix), Qiagen (Type-it CNV SYBR Green PCR Kits), Roche Applied Science (FastStart SYBR Green Master), Sigma-Aldrich (KiCqStart® SYBR® Green qPCR ReadyMix™), and Thermo Scientific (e.g., Luminaris Color HiGreen High ROX qPCR Master Mix), among others.
Probe-based reactions
The probe-based approach uses a sequence-specific, fluorescent oligonucleotide probe to add specificity to a qPCR reaction. The most common form of this approach uses Life Technologies’ TaqMan® chemistry, which uses a DNA probe containing a fluorophore on the 5’ end and a fluorescent quencher on the 3’ end. As PCR proceeds, the probe hybridizes between the forward and reverse primers. When the polymerase reaches the probe, its 5’-3’ exonuclease activity liberates the fluorophore, freeing it from the quencher and inducing fluorescence.
Unlike dye-based qPCR, probe-based methods signal (in theory) only the targeted sequence. They also can be multiplexed, meaning multiple reactions can be run in parallel in a single tube.
Probe-based master mixes are available from Agilent (Brilliant III Ultra-Fast QPCR & QRT-PCR Master Mixes), Bio-Rad Laboratories (e.g., SsoFast Probes Supermix), Life Technologies (e.g., TaqMan Fast Advanced Master Mix), Promega (e.g., GoTaq Probe qPCR Master Mix), Qiagen (Type-it CNV Probe PCR +qC Kit), Roche Applied Science (FastStart TaqMan Probe Master), and Thermo Fisher Scientific (DyNAmo ColorFlash Probe qPCR Kit), among others.
Nolan, who coauthored a 2009 report outlining how best to run, report, and analyze qPCR data [1], estimates that 80% to 90% of qPCR users employ either SYBR Green or TaqMan, with users split 50/50 between the two approaches. The remainder mostly rely on variants of the two, she says, such as alternative probe-based strategies like Molecular Beacons and Scorpion® primers.
A Molecular Beacon is a DNA hairpin probe capped at either end with a fluorophore and quencher. As with TaqMan, the probe is complementary to a target sequence between the amplification primers. During annealing, this structure unfolds and binds to its target sequence, thereby separating fluorophore from quencher and producing a fluorescent signal proportional to the abundance of the target site. A Scorpion probe is a fusion of a Molecular Beacon with a PCR primer; extension of the primer during amplification creates a region of complementarity for the probe, which then unfolds and binds its target, again inducing fluorescence.
Promega offers a third, completely different sort of qPCR chemistry called Plexor®. Unlike other qPCR strategies, which record an increase in fluorescence, Plexor looks for the loss of fluorescence, explains Gabriela Saldanha, Strategic Marketing Manager at Promega.
In Plexor, one primer is labeled with a modified nucleotide (“iso-dC”) linked to a fluorophore. As amplification proceeds, the DNA polymerase pairs that base with another modified nucleotide, Dadcyl-iso-dGTP, which quenches the fluorophore.
Plexor offers two advantages, Saldanha says. The first is that it combines the sequence specificity of the probe-based strategies, with the melt curves of dye chemistry. “You can verify the specificity of your target of your amplification,” she says. But in addition, Plexor probes can also be multiplexed by coupling different dyes to each primer pair. Saldanha says the company has multiplexed up to six reactions at once using that strategy, though she adds, “That would really depend on really good primer design.”
Quantifying gene expression
qPCR applications include everything from SNP genotyping and copy-number detection to pathogen detection. But the most common application, Saldanha says, is gene expression analysis.
Gene expression analysis involves quantifying copies of a specific RNA in a sample, whether that be an mRNA, microRNA, or long noncoding transcript. Because RNA cannot be PCR-amplified, this application requires the conversion of RNA into a DNA copy that can be amplified – that is, cDNA synthesis.
cDNA synthesis/qPCR kits are available in one- and two-step formats, Saldanha says. One-step kits combine cDNA synthesis and qPCR in a single step, whereas two-step kits separate the two reactions. The former is advantageous in terms of speed and automation, whereas the latter is more efficient and allows users to retain cDNA samples and test a single RNA sample for multiple transcripts.
“Two-step [qPCR] has an advantage, because you are being more efficient at creating cDNA and in qPCR,” Saldanha says.
RT-qPCR kits are widely available, including from Agilent, Bio-Rad Laboratories, Life Technologies, Promega, Roche Applied Science, and Qiagen.
Sample considerations
When it comes to qPCR, it takes a good nucleic acid prep to get good data. Unsurprisingly, not all samples are equivalent in this regard. “Environmental” samples, such as dirt samples, can be especially difficult, says Saldanha, as they often contain compounds that can inhibit PCR reactions. Also difficult because of inhibitory compounds are plant samples, says Karudapuram.
FFPE samples also present difficulties, not just because of potential inhibitors, but because recovery is often low and nucleic acids can be degraded. “In that case, the sensitivity comes into play,” Karudapuram says, “because we’re talking about very small amounts of template. So in addition to having purer extraction of nucleic acid material, you also need a master mix that can amplify from small amounts of starting template.”
In such cases, consider nucleic acid extraction kits optimized for your particular application, such as Promega’s ReliaPrep™ FFPE RNA Miniprep System or Life Technologies’ Plant RNA Reagent.
Also consider the primers you use to amplify your template. “While it’s good to design your own assay, for greater standardization, it makes more sense for everybody to use the same assays for the same gene,” says Nolan.
Towards that goal, several companies now offer libraries of pre-designed qPCR assays. Sigma-Aldrich has prepared libraries for human, mouse, rat, and zebrafish under its KiCqStart brand, and other vendors also offer similar tools including Qiagen, Life Technologies, Integrated DNA Technologies, and Thermo Fisher Scientific.
qPCR instruments
To record qPCR data, you’ll need a qPCR instrument, which is essentially a combination PCR thermal cycler and fluorescence reader. Such instruments are available from such vendors as Agilent (Mx3000P qPCR System), Bio-Rad Laboratories (e.g., CFX96 Touch™ Real-Time PCR Detection System), Illumina (Eco Real-Time PCR System), Life Technologies (e.g., QuantStudio™ 12K Flex System), Qiagen (Rotor-Gene Q), Roche Applied Science (LightCycler® systems), and Thermo Scientific (the PikoReal Real-Time PCR System), among others.
Digital PCR
Clearly, qPCR is a robust and mature technology. Yet it does still struggle with some quantification applications, says Nolan, especially when the absolute differences between two samples are relatively small. For instance, distinguishing two copies of a gene from three copies can be challenging for qPCR, as is, say, prenatal genetic diagnosis from maternal blood samples.
This is where digital PCR comes in, Nolan says. In digital PCR, a sample is diluted into multiple partitions on a plate such that each partition contains, on average, zero or one copies of the template. Then a qPCR reaction is run in each well, and the positive wells counted. From those data, it is possible to determine the absolute number of template molecules in a sample. “You can differentiate, say, 100 copies from 130 copies,” Nolan says, adding: “It’s really, really, really clever.”
Digital PCR tools are available from Bio-Rad, Fluidigm, Life Technologies, and RainDance Technologies, among others.
Long story short: If you need to quantify nucleic acids, you’ve got plenty of options.
Reference
[1] Bustin, SA, et al., “The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments,” Clinical Chemistry 55:4 (2009), doi:10.1373/clinchem.2008.112797
The image at the top of the page is from Bio-Rad Laboratories.