Whatever the technique, it is essential to have the correct experimental setup to let you verify that you are measuring what you think you are measuring. And PCR is no different – you can’t conclude anything concrete from your results unless you have the correct controls in place.
Ian Kavanagh of Thermo Scientific has a very illuminating the topic of controls and standard curves in PCR lined up for today’s live seminar here on Bitesize Bio (click here to reserve your place) but in this article, I’ll give a quick run-through of the basic controls you should be including in your PCR reactions.
No template controls (NTC)
Negative controls that contain everything but the template DNA are essential for detecting contamination or non-specific amplification in your reaction. Correctly used, NTCs can not only alert you to the presence of a contaminating amplicon, but can also help you identify the source of the contamination. For more details of that, see Ian’s seminar.
Please never be tempted to miss out the NTC. They are really easy to set up, but you are just asking for a beautifully amplified non-specific product to appear in the right place on your gel if you miss it out. But don’t just take my word for it – the MIQE guidelines (we have a seminar on those too!) advise that NTC’s should be considered an essential part of every PCR reaction.
No Enzyme Controls (NEC)
“No enzyme” controls are required when using PCR to quantify RNA, which involves the PCR amplification of a cDNA synthesised from the target RNA sequence using reverse transcriptase. The reverse transcriptase is omitted from the NEC (or the “no-RT” control) and this can be used to determine that any amplification that occurs in the sample is derived from the synthesised cDNA and not genomic DNA or other amplicon contamination.
If the NEC shows that contamination is present, then cleaning up the sample or using intron-targeted primers that will only bind to the RNA can solve the problem.
Positive controls are needed for the verification of negative amplification results and the positive control reaction should contain the same components as the sample but include a template that is certain to amplify if the reaction goes as planned.
This could be an external positive control, which is a separate sample containing the control template. Such external control reactions can help detect when a reaction fails due to cycler or reaction component problems or when an inhibitor is suppressing the reaction.
Alternatively, you could use an internal positive control (IPC). To run a reaction with an IPC, the template and primers for the control target are included in the reaction along with those for the target of interest. The control target should of course be easily distinguished (by electrophoretic migration or Tm) from the target of interest. As well as having the advantage that of not requiring a separate reaction, IPCs are useful because they can pinpoint problems that are intrinsic to the sample reaction.
There are so many things that can make a PCR reaction go wrong. So habitually including a positive control is certain to save you a fair amount of head-scratching during the course of your career by pinpointing those times when one of the reaction components is causing you problems.
More detailed information on controls in PCR can be obtained in Ian Kavanagh’s seminar, which airs live at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET TODAY. There are still some places left and you can secure yours by clicking here.