Top Tips for Real-Time qPCR Experiments – Your Questions Answered
In a RT-qPCR webinar masterclass – now available on demand – expert Melanie Jahn shares her top tips for optimization of your assay
30 Oct 2018
Expert insights
The polymerase chain reaction, PCR, is a common technique in molecular biology that can be used quantitatively or semi-quantitatively to monitor the presence and/or quantity of DNA in a sample. Uses involve diagnostics, microbiology research and in measuring gene transfection or modification efficiency.
The real time element of more advanced assays offers many advantages to researchers, including more accurate quantification during exponential phases, elimination of post-PCR electrophoresis and a reduced possibility of sample contamination. All this and more is addressed during a webinar presentation by Melanie Jahn, of Analytik Jena, which is now available to view on demand. We’ve summarized Jahn’s top tips from the audience question and answer session below.
Q: Should I use qualitative or quantitative PCR?
MJ: What you decide to use in your lab is a matter of what you want from your result. A qualitative result is generally a yes or no statement, for example, contamination of a pathogen or not. A quantitative result is concerned with concentration determination: determining unknown quantities of DNA in a sample.
Q: How many standards would you suggest for a proper standard curve?
MJ: I would suggest performing three as minimum. Obviously, the more standards the better the curve, so more is always better – six is ideal.
Q: What is the best way to set a threshold for your experiment?
MJ: The setting of the threshold is the most sensitive part of the experiment – a lot of errors can happen here. I would suggest having a look at the logarithmic part of the curve and then set the threshold within the logarithmic phase, so it’s at the beginning of the exponential phase of the qPCR. It is important to set it in the same way for every repeat experiment, so you can compare experiments which each other.
Q: What is the best way to avoid inhibitors affecting your results?
MJ: Inhibitors have a big influence on the result. To avoid them I would recommend performing the DNA or RNA extraction thoroughly – for example, removing the ethanol within your extraction methods and performing a dilution of your extracted nucleic acid. A 1:10 dilution can help to avoid these inhibitors.
Q: Is there a difference between PCR grade water and molecular biology grade water?
MJ: No, in fact there isn’t any difference – just the naming used by different suppliers.
Q: In your talk, you mentioned that one factor for poor PCR efficiency was poor primer design. How do I measure this?
MJ: Unfortunately, you cannot test this – the only thing you can do is design new primers and compare the efficiency of these, with the old.