qPCR is an extremely sensitive technique that allows researchers to produce millions of copies of a specific DNA sequence from a few initial copies. While this sensitivity is very useful, it comes with a down-side. If DNA fragments from the lab environment, such as a DNA template amplified in a previous qPCR experiment, enter the qPCR reaction or reagents, even in small quantities, they can be amplified during the reaction. This contamination and non-specific amplification can cause misleading results, such as false positives. If you are new to conducting qPCR experiments, it can be difficult to spot contamination issues, and to avoid or reduce contamination risks.
DNA Contamination can not be reduced or removed once it has occurred. Therefore to prevent contamination, it is essential adopting best laboratory practices when performing qPCR experiments, and introducing and enforcing proper laboratory or institution-wide procedures. In this article, we discuss the different sources of qPCR contamination, determining if your experiments suffer from contamination issues, and basic best practices and procedures for avoiding contamination.
How can you tell if contamination is an issue in your qPCR experiment? One of the most common ways to monitor for contamination is to use “no template controls” (NTCs). In a standard 96-well plate qPCR setup, NTC wells contain all the qPCR reaction components components such as primers, reagents etc., with the exception of the DNA template . If the NTC wells are contamination-free, you should not observe any amplification in these wells following the thermocycling steps. If amplification is observed in the NTC wells, then it may be possible to discern the cause of the contamination, which could help you decide on an appropriate response.
If, for example, one of your reagents has become contaminated, then you would expect to see amplification in each NTC well containing that reagent, at a similar Ct value in each well. In such a scenario, try replacing reagents you suspect are contaminated. The observed contamination could also be random, e.g. an aerosolized DNA template in the lab environment could drift into wells on your qPCR plate prior to thermalcycling. In this case, you would expect to see amplification in only some NTC wells with different Ct values for each contaminated NTC well . In both instances, you may need to review and improve your general laboratory procedures and practices to reduce and avoid contamination.
Physically separating qPCR processes
You can use a variety of simple, practical approaches to avoid contamination. Begin by establishing separate, dedicated areas for different processes in the qPCR workflow, e.g. sample preparation, qPCR setup, qPCR amplification, analysis of qPCR products. How you do this will depend on the laboratory space and equipment available to you, but at a minimum, implement separate pre- and post-amplification areas.
One of the major qPCR contamination sources is amplification carryover contamination. During the qPCR amplification process, millions of copies of the DNA template are produced. When you open a tube or plate containing the amplified product, significant quantities of the product can be aerosolized and easily dispersed into the lab environment. If this aerosolized DNA template contaminates a reagent, Master Mix or qPCR reaction in subsequent qPCR experiments, it can easily amplify.
Therefore, it is best practice to keep pre- and post-amplification areas separate, and ideally in different rooms with completely independent laboratory equipment such as pipettes, centrifuges and vortexers. Ensure each area has its own protective equipment, such as gloves and lab coats, and a dedicated supply of consumables. Ideally, these rooms should not be supplied by the same ventilation/ducting system. You should also maintain a one-way workflow between these different areas, where researchers who have been working in a post-amplification area do not enter a pre-amplification area on the same day. If you need to go from a pre- to a post-amplification area, you should change your gloves and lab coat . It is important to de-contaminate anything that is used in post-PCR area before bringing it back to pre-PCR area. Beware that it is also possible to transmit contamination via jewelry, cell phone, even hair, and not just through unclean gloves or labcoats.
Personal protective equipment, liquid handling and storage
Be aware of when your gloves may have become contaminated, e.g. through exposure to a splashed reagent. Changing your gloves could prevent you from contaminating the surrounding work surfaces, plasticware and equipment. Open tubes carefully to avoid splashing or spraying their contents. Keep samples and reactions capped/covered as often as possible, and dispose them in a safe, contained place after use. Use a positive-displacement pipette and aerosol-resistant filtered pipette tips, and ensure that your pipetting technique is not causing unnecessary splashing or spraying. This could reduce aerosol formation in your samples or reagents .
Finally, store samples separately from kits and reagents in pre-PCR areas, store PCR products in post-PCR areas. If possible, you should aliquot reagents such as primers and probes, into volumes suitable for a single experiment, to prevent repeated opening and freeze-thawing of stock solutions.
Surface and equipment decontamination
We have discussed the use of physical separation and personal protective techniques to reduce contamination, but it is also good practice to regularly decontaminate surfaces and equipment that are utilized for preparing qPCR reactions. Centrifuges and vortexes are prone to contamination. You can significantly reduce contamination of work surfaces and equipment by cleaning them before and after qPCR using 70% ethanol.. Thorough cleaning is particularly important after a spill. Use a 10–15% bleach solution (sodium hypochlorite) for the best results, and remember to use gloves and eye protection while cleaning with the diluted bleach solution. Make fresh dilutions of bleach as often as possible (at least every week or two), as it is unstable and may not be effective if stored for long periods. Allow the bleach to work on the surface for 10 to 15 minutes before wiping the area or equipment down with de-ionised water.
The measures we have discussed so far, if applied diligently, can significantly reduce the chances of contamination. The implementation of best laboratory practices can be complemented through the use of certain reagents that allow you to eliminate some types of contamination from your samples and reaction mixes prior to thermocycling.
An enzyme called uracil-N-glycosylase (UNG) present in certain qPCR Master Mix formulations removes carryover amplification contamination from your reactions. UNG destroys carryover amplification contamination from previously amplified templates, selectively targeting templates that contain uracil instead of thymine. This technique requires that you use a deoxynucleotide (dNTP) mix that contains uracil instead of thymine when performing your qPCR amplifications; this way, all of your amplification products will contain uracil.
The enzyme is active at room temperature, therefore it can be incubated with your amplification mixture to inactivate any sources of carryover contamination. Once thermocycling is initiated, the high temperatures inactivate the UNG, thus preventing the enzyme from affecting the newly generated amplification products, even if they contain uracil. The UNG method works best with thymine-rich amplification products and is not as effective with guanine/cytosine-rich amplification products. UNG is not effective for sources of DNA contamination, other than uracil-containing amplification products from previous qPCR experiments .
This article has covered a variety of approaches to reduce or avoid contamination in your qPCR experiments. Basic prevention procedures in particular, such as routinely decontaminating surfaces and equipment, and physically separating pre- and post-amplification areas and equipment, will go a long way toward keeping your experiments contamination-free.
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3. Amplification of the No Template Control (NTC)
4. TaqMan Gene Expression Assays protocol
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