Quantitative polymerase chain reaction (qPCR) is a staple in any lab dealing with nucleic acids, such as cancer research labs (Check out our top product choices for cancer research here) capable of producing amazingly accurate measurements. However, many scientists still find themselves having trouble producing reliable and reproducible results every time.
Here are a few common mistakes that could be the cause of your qPCR woes, and our suggestions of how to avoid them.
1. Your Sample Has Been Contaminated
It happens. DNA is everywhere, and unless you take proper precautions it will end up in your sample too. Furthermore, other contaminants such as hemoglobin, ethanol and phenol can inhibit PCR.
There are many points at which contamination can enter your samples. Here are a few tips for minimizing your risks of a contaminated sample.
- The first step is a clean workspace. A PCR bench (AKA clean benches, laminar flow hoods etc.) is a great start. PCR workstations prevent background and cross contamination by keeping all contaminants in the air away from samples.
- All surfaces in the PCR area should be routinely decontaminated to prevent cross contamination, using a DNA decontamination solution that destroys DNA.
- Have several sets of pipettes on hand and keep pipettes for different workflow steps separate.
- Consider including a minus-reverse transcriptase control (or "No Amplification Control" / NAC) in qRT-PCR experiments. This is usually a simulated reverse transcription containing all the RT-PCR reagents apart from reverse transcriptase. If a signal is seen in the NAC, it likely means that you have a DNA contamination in your sample.
- You can also use a spectrophotometer to give you an idea of your DNA concentration and quality. The ratio of the absorbance at 260 and 280 nm (A260/280) should be in the range of 1.8-2.0. If it deviates, your samples may have been contaminated with salts, solvents, or protein. If you do have contamination, run a cleanup protocol, such as a clean-up kit.
2. Your Sample Has Degraded
Degraded RNA looks like a smear on an agarose gel and doesn’t form recognizable ribosomal RNA bands. But how did this happen? Well, there are several things to consider if you want to keep your sample in good condition to run it through a PCR.
- Store your samples properly. It cannot be overstated how important it is to invest in proper storage for your samples. If they are sensitive to any environmental factors, you need to be 100% certain that these factors are under control. Storage of samples includes anything from the equipment, to the vials, and the buffer, and you need a system to monitor if your samples are being threatened by a change in environment. Especially if your sample is rare, difficult to obtain or expensive, it makes no sense not to invest in taking proper care of it and use an RNA-preserving buffer. We cannot stress this enough. For best results, you want your samples to be as fresh as possible, so avoid putting them through numerous thaw/freeze cycles.
- Your sample may have been contaminated with nuclease enzymes. Always make sure to clean your workspace properly using RNase inactivating solution. You can also add a reagent such as RNAsecure™ to your solution to remove RNase contaminant in your sample.
- Most commercial PCR kits contain an RNase inhibitor, and for good reason. If you’re still using a kit that doesn’t contain one, it may be time to switch to a kit that does.
- Finally, it’s important to only ever use nuclease-free water. You can get it ready-to-use or make your own in the lab.
3. Not Having the Right Primer and Probe Design
- For best results, it’s highly recommended that you use a primer design software. NCBI offers a free primer design platform that you can take a look at.
- When designing amplicons in eukaryotic targets, choose PCR primers that span at least one exon-exon junction in the target mRNA to prevent amplification of the target from contaminating genomic DNA.
- Something else to keep in mind with primer design are the primary and secondary structures that can impact the reaction, and a software can help you work around that.
- Ideally, you’d want to avoid regions with a lot of base-pair repetitions, but this is obviously not always an option.
- Regions that consist of more than 60% of GC can be particularly tricky, because they are not only quite thermostable, but their “bendability” means that they have a tendency to form secondary structures, like hairpins. Luckily, there are commercially available additives that can help you solve this problem, such as the correct master mix or polymerase.
4. Not Using Master Mixes
Which brings us to the next point.
qRT-are extremely sensitive and efficient tools for amplifying RNA, which is great, until it also amplifies errors. Of course, variability between wells and samples should be kept to a minimum, and reproducibility to a maximum. This is where master mixes come in. To further reduce well-to-well variation, a reference dye such as ROX can be added to the master mix.
5. User Error
No one is perfect, and especially when it comes to fiddly and often repetitive work, mistakes will happen. There are a few common errors that can happen, especially when you are tired or hungry.
You forgot to add something
Getting strange results? No results at all? It's possible that you have simply forgotten to add something important to your reaction. Go through your steps again and see if you may have missed something.
The wrong PCR conditions used
Many machines give you the option to save your personal settings. This is great if you have several groups with different protocols working on the same machine and makes it much faster to set up your experiment every time, but of course it can prove a pitfall if you happen to select the wrong save setting for your reaction. Always double-check that you have the correct setting selected before you start.
Too high annealing temperature used
Sometimes figuring out the best temperature for your reaction can be tricky. One great way to find the best temperature is using a PCR machine that has a gradient functionality. You can perform a gradient PCR to test a variety of annealing temperatures at once, which you can then use to compare and pick the temperature which gives you the brightest desired band.
Forgot to add dye to the agarose gel
If you run a gel and see a whole lot of nothing in your results, you may have forgotten to add a dye altogether. One convenient way around this would be to get a pre-cast gel with the dye already in it.
On top of these tools and tips to help make your life in the lab easier, and to reduce the chances of these types of user errors we also recommend that you take regular breaks, get enough sleep, food and vitamin D.