How to Avoid Contamination in Your qPCR Experiments

Great Tips for how to make the best out of your experiments

qPCR, with its high sensitivity, allows researchers to produce millions of copies of a specific DNA sequence from only a few initial copies. While this sensitivity is very useful, it comes with a necessity to avoid contamination of the reaction.
Contamination and non-specific amplification can cause misleading results, such as false positives and masking changes between the control sample and the tested sample. When working with plasmids, the problem of contaminations becomes even more severe and difficult to deal with.
DNA Contamination cannot be reduced or removed once occurred. Therefore, it is essential to adopt adequate laboratory practices when performing qPCR experiments.

Identifying contamination
How do we know if we have a contamination is our qPCR experiment?
One of the most common ways to monitor for contamination is to use “no template controls” (NTCs). NTC wells contain all the qPCR reaction components such as primers, master mix and water, except for the DNA template. If amplification is observed in the NTC wells, it is possible there is a contamination in one of the reaction components.

How to avoid contamination 

►Laboratory Construction– Contamination prevention starts with the division of the qPCR laboratory working space. At minimum, two areas should be designated for PCR testing, ideally in different rooms with completely independent laboratory equipment such as pipettes, centrifuges and vortexes.
  • Pre-PCR: Optimally, this room should be further divided into two areas-
  ° PCR master mix preparation- Ideally with a dedicated set of pipettes.
  ° sample preparation, extraction and addition to master mix.
  • Post-PCR: this room (or bench) should be physically separated from the pre-PCR room, designated for post amplification steps and analysis. In this area you will want to locate the qPCR instrument and perform all steps which require open tubes manipulation after PCR amplification.
** Ideally, these two rooms should have completely independent laboratory equipment such as pipettes, centrifuges and vortexes. Ensure each area has its own protective equipment, such as gloves, lab coats and a dedicated supply of consumables. Ideally, these rooms should be ventilated by separate systems as well. 
 
►Unidirectional Workflow – A “one-way” workflow between the different areas i.e. Pre-PCR à Post-PCR is an important rule.  This means that consumables and PPE (lab coats, gloves, goggles, etc.) that have been introduced into the post-PCR room would never be placed back in the pre-PCR room without thorough decontamination. Be aware that it is also possible to transmit contamination via jewelry, cellphone and even hair. 
 
►Reagent handling – 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 and master mixes into volumes suitable for a single experiment, to prevent repeated opening and freeze-thawing of stock solutions.
 
►Pipetting – 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. Open and close all sample tubes and reaction plates carefully so samples wont splash out. Spinning the tubes/plates before opening, can prevent aerosols when opening tubes.
 
►Frequently Changing Gloves – 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.
 
►Surface and equipment decontamination – Regularly decontaminate surfaces and equipment that are utilized for preparing qPCR reactions. This applies to all work surfaces including bench tops, pipettors, fridge/freezer handles, centrifuges, vortexes and any other touch points. Use fresh 10–15% bleach solution (0.5-1% sodium hypochlorite), Allow the bleach to work on the surface for 10 to 15 minutes before wiping the area or equipment down with de-ionised water. This can be followed by a 70% alcohol dampened paper towel to help quickly dry the surfaces. 
 
►Sample decontamination – in order to prevent PCR product contamination, ThermoFisher use master mixes with uracil-N-glycosylase (UNG) enzyme that removes carryover amplification contamination from previously amplified templates. This technique requires using a deoxynucleotide (dNTP) mix that contains uracil instead of thymine when performing your qPCR amplifications. This way, all the amplification products will contain uracil and will be digested in the beginning of the qPCR reaction. 

References:

1. Aslanzadeh J (2004) Preventing PCR amplification carryover contamination in a clinical laboratory. Ann Clin Lab Sci 34(4):389–96.
2. Nolan T, Huggett J, Sanchez E (2013) Good practice guide for the application of quantitative PCR (qPCR), LGC.