Within a decade of its discovery in 1983, Real-Time PCR—also called quantitative polymerase chain reaction (qPCR)— evolved into one of the most powerful and sensitive gene analysis techniques available. Real-time PCR measures PCR amplification as it occurs, so that it is possible to determine the starting concentration of nucleic acid. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA.
In traditional PCR, which is based on end-point detection, results are collected after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid. Every real-time PCR contains a fluorescent reporter molecule—a TaqMan® probe or SYBR® Green dye, for example—to monitor the accumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore.
PCR reagents are sold individually or as “master mixes.” The most purchased qPCR assay reagent format among respondents was individual reaction components sold separately in different tubes. The separate purchase route allows end-users to fine-tune PCR reactions with favored (or less expensive) reagents, but this requires a fair degree of expertise. While more expensive, master mixes are a good deal because they contain all PCR reagents except the primers and, of course, the template — as a result, they save the lab time. Master mixes are gaining in popularity, especially among non-expert users.
Experts suggest that if you are operating small, then you may want to buy individual reagents and save money by making your own master mixes. However, if you are brand-new to the game, it’s highly recommended that you start with a kit, because you don’t want something going wrong because of the reagents.
Average reagent cost per qPCR well. (reagents only)
|Less than US$ 0.25 per well||10%|
|Between US$ 0.25 - 0.50 per well||17%|
|Between US$ 0.51-0.75 per well||7%|
|Between US$ 0.76 – 1.00 per well||17%|
|Between US$ 1.01 - 1.50 per well||10%|
|Over US$ 1.50 per well||12%|
Advantages of real-time PCR include:
- Generation of accurate quantitative data
- Increased dynamic range of detection
- Elimination of post-PCR processing
- Detection down to one copy
- Increased precision to detect smaller fold changes
- Increased throughput
Used for a broad range of applications, the most common uses of quantitative PCR are gene expression analysis, genotyping, SNP analysis, pathogen detection, drug target validation and for measuring RNA interference.
|Copy number analysis||12%|
|Microarray/miRNA / RNAi validation||10%|
The number one factor in buying qPCR reagents is performance / quality of reagent (speed of analysis, fidelity of DNA-copying). Experts suggest first talking to vendors and getting them to send your lab a small aliquot to try. Then do some side-by-side comparisons with your old and new reagents, using the standards that you use for all your assays, and see if the new reagent works better or at least as well. If it has a better price, then it’s worth considering. Experts also recommend that labs should always determine the exact amount of what is in the tube by looking at the optical density of the primers and probes and use what’s on the label only as a guide. There are also some problems with contamination. For instance, every primer and probe has some slight DNA contamination, which you can pick up with assays that are very sensitive. Thirty-nine percent of the respondents purchase kits and reagents for qPCR from both their instrument’s manufacturer and reagent companies, while 38 percent purchase just from reagent companies, and 24 percent buy only from instrument manufacturers
|Better quality reagents||93%|
|Better technical support/customer service||43%|
|Wider selection of novel kits||17%|
Singleplex is a reaction in which a single target is amplified in the reaction tube or well. Running both assays in a single tube reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes.
Multiplexing is generally more complicated than simply adding additional primers and probes to a singleplex qPCR. Additional consideration must be given to the selection of primers, probes, cycling conditions, and reaction formulation. Multiplex qPCR is a technique that simultaneously amplifies two or more target sequences in a single reaction. Analysis of multiple targets per sample allows for conservation of limited starting materials, the ability to run an internal control, and increased sample throughput. Multiplex qPCR is not possible when using SYBR Green chemistry.
|Currently Doing||Planning to Do|
|Single-Plex qPCR (using just one channel)||56%||28%|
Data quality expectations were rated as the factor most influencing respondents’ purchase of a qPCR instrument. Experts advise that if you are buying a new instrument, you can get the manufacturer to bring the machine in for a week or so, to see how it works. Sometimes, people buy new PCR machines with a certain project in mind, but what you really have to do is think ahead. Comments from some respondents were: if you buy an inexpensive instrument or don’t buy the various software modules, then you may not be able to do certain things like run 384-well plates, multiplex, or HRM. Spend a little more of your project dollars to get a real-time PCR machine that has all the flexibility you need in the thermal cycling programming, detection formats, and data analysis.
Forty percent of respondents have selected automation of qPCR assays as an important factor. With continuous improvements in automated platforms, automation of qPCR assays is expected to increase in the future and have the greatest impact in advancing qPCR
Top ten factors in the decision to purchase a qPCR instrument
|Consistent quality of data||93%|
|Service and support||88%|
|Ease of use||86%|
|User-friendly controls/software interface||84%|
|Linear dynamic range||61%|
Completed Surveys: 194
For more information on qPCR, visit www.labmanager.com/qPCR