Since 5 years we use quantitative PCR (real-time PCR, qPCR) analysis in my lab. And now it seems that enough experience is collected to write a brief report and some recommendations for those who plan to establish quantitative PCR (real-time PCR, qPCR) technology or experiences some difficulties.

I do not think that the principle of quantitative PCR (real-time PCR, qPCR) has to be explained in detail; therefore I believe one sentence will be ample. Real-time PCR is a PCR reaction set in a way that the amount of amplified product is measured after every cycle.

There are several ways to measure the amount of product synthesized, and every company that sells reagents for real-time PCR will explain you that their product is the very best one. However there are 2 major classes of detection systems: 1) intercalating dyes and 2) probes.

The first one uses a fluorescent dye that starts to fluorescent when incorporated in double stranded DNA. Example of such a dye is SYBR green. Advantage of the method is its price. Disadvantage – low specificity, since everything double stranded will be stained. I used SYBR green approach for a while and even produced good results. But there were also some funny results. Once I used genomic DNA as a template and at the end of the reaction found the reduction of fluorescence, and not an increase, what you would expect when performing PCR. The reason for this was simple. I used too much of the template. My entire genomic DNA was nicely stained and cycle by cycle more DNA was denatured and stayed denatured, so the amount of fluorescence was reduced after every cycle.

Second approach uses and additional primer that binds PCR product and either gets degraded by Taq polymerase (Taq has this activity if incubated at 60°C), or stats to fluorescent for some other reason (these are all molecular beacons and so on). This way of detecting a PCR product is much more specific, but significantly more expensive. This approach has theoretically one more advantage. You can combine 2 or more PCR reaction in one tube if you use different dyes for detection. This is so-called multiplex real-time PCR. I wrote theoretically, since every time we tried to establish such a reaction we failed. Again, I don’t want to give you explanations, only one tip. You should try to establish a multiplex PCR only if you have a large number or samples you have to analyze (with large number I mean hundreds and thousands). Usually it does not make sense to spend time and reagents optimizing multiplex reaction if you only have to analyse <100 samples.

Which approach to choose? It is again a question of your time and money. If you have time to play with SYBR green and want to save money – try this method. If you can invest in speed and specificity – use probes. And again, if using probes you have 2 options: 1) purchase ready optimized primer/probe mix or 2) design and optimize probe/primer mix yourself. Second option requires some optimization, but it is roughly 10 times cheaper than ready mix.

Hardware

To perform real-time PCR you need a special PCR cycler that is capable of reading fluorescence after every cycle. There are quite a few instruments on the market that became affordable now. They utilize all possible combinations of excitation and detection systems. Following technologies were used in instruments I read about: halogen lamps, LEDs and lasers for excitation and  CCD cameras and PMTs for detection.

The very first instrument that was purchased in our institute was ABI Prism 7000. This instrument uses halogen lamp that simply hangs over the plate and a cooled CCD camera that made a snapshot after every cycle.

At the moment we use Mx3005P instrument from Stratagene. It uses focused light from halogen lamp for excitation and PMT for detection.

To be continued... You can post you questions in the forum.