No, it is not possible to run them in a single run. You have to start two runs: one for your housekeeping gene and the other one at the perfect temperature for your gene of interest.

So normalization of target gene will be done after seperate runs? But when i looked at the manual of qPCR machine(Stratagene) it says run normalizer and target gene in the same run.

I also got some advises like doing the qpcr run by using the lowest annealing temperatures and the other one was like doing qpcr with 20cycles with one Ta and the other 20steps with other Ta. What do you think about this ideas?

thanks

Hi,

you have to run the target gene and the reference gene in one run. Otherwise normalization wouldn´t make sense because you do it to compensate the variations in pre-pcr-steps and the pcr itself. The last one is not possible if you run the target gene seperated from the reference gene.

In your case it is not possible because of the different annealing temperatures. You really have to redesign your primers. Try to find some with an annealing temperature near 60°C. Don´t forget that your primers should run over an exon exon junction to be transcript specific. Also the product lenght should not be >200 bp to make sure the pcr efficiency is near 2.0 = 100 %. GC content between 40 and 60%, primer length ~20bp, GC contente in the last 5 bp not over 2-3. Also your primers should not run over repetitive sequences and snp´s. Make also sure (for example with primer stats) that your primer pairs don´t tend to form dimers or hair pins.

If it is not possible to design a good primer which spans an exon exon junction, design it in the exons instead but make sure that you total RNA is gDNA free prior to RT.

Especially for the reference primers you really have to make sure too find some good ones. Normally good reference primers work at different annealing temperatures if they are near 60°C. If you can´t manage this you have to design functioning reference primers for every target gene with a different annealing temperature.

If you have any more questions on the matter you can also contact me at [email protected]

I just finished a successfull RT-qPCR-project and are pretty much up to date with the matter.

I know it sounds not funny. But primer design for RT-qPCR experiments is not as easy as primer design for the amplification of gDNA. You really have to spend some time on it. It is one of the critical steps in planing an experiment. Bad primers lead to non reproducible results.

Have fun in the lab anyway and relax. It takes some time and experience to optimize a good RT-qPCR experiment.

Best Regards

Daniel

I have to disagree with Daniel since in most of the cases it will not be doable practically. I am running everytime a standard curve in addition to my normal sample, as long as the curve is telling me roughly 100% efficiency it is fine to run them separate. As you can imagine you can never run all your genes you want to check in one run even when they will have the same annealing temperature since you will have limited space in the machine, specifically when you are running triplicates. Therefore, a standard curve in each run with the primers and different concentrations of template is going to help you, since you can calculate the values of the standard curve into the normalization. I hope that helps!

Hi Nicole,

I made the experience that it is always possible to design your target/reference primers to match each others annealing temperatures. I never had to run my reference primers in different reaction-setups. To be honest I never heard of anyone who did this. For me it would not make much sense because you have much more bias in your results afterwards.

Don´t get me wrong. I don´t want to attack your opinion. Different work groups in different labs make different experiences. But for my understanding, the relative quantification using one or more reference primers just make sense if they are used in the same setup as the target primers. I mean it is not just about efficiency but also variation due to pipetting and pcr at different temperatures. So one should always try to optimize the primers so they could be used together in one run.

Best Regards

Daniel

I completely understand your opinion and I agree that the temperature should be in a similar range. Nevertheless, if you would have 20 samples and want to run triplicates for two different housekeeping genes and probably 5 different genes of interest how do you do that if you just have a 96 well plate machine? You have to take into account you want to compare all 20 samples (for example 20 different tumor samples) with each other.

I can see your point. But to completely seperate the target amplification from the reference amplification might not be the optimal way. I would try to match the anealing temperatures and subsequently run the reference gene and the target gene always on one plate. Of course this would mean that you have to distribute the samples on severeal plates. Isn´t that state of the art anyway?

No, it is not the state of the art at least for tumor samples for example. All the people working with human tumor samples I know, are running the control gene completely in one run of all samples as long as possible otherwise if you have too many prepare mastermix at the same time and then run them in two runs (with in both runs 2 samples being the same). Afterwards, you can check at any time each of your genes of interest the same way. Just try it (also to Pinar): run the same samples twice with the same mastermix in two separated runs of the machine. They will be very similar. I hope that helps!

Well,

that sounds interesting. Never heard of it. Thanks for the heads up. I will keep it in mind.

People specifically with primary human samples do not want to rerun everytime the control gene due to limited amount of template. Furthermore, they are thinking of an additional gene to check one year later after they did enough cell culture to identify that one as a putative biomarker and then they are going back to check that one in all samples.

Moved to quantitative PCR topic. Please post in the right topic in the future. Thanks,

Axel

I am not so into the clinical use of qPCR. My field is more animal genetics. You guys have to deal with larger sample sizes and hence are in much more need to improve the throughput.

Anyway thanks again. It is always nice to learn some more on the whole methodology.

Theoretically you have to redesign your primers. The Tm of your housekeeping gene and the GOI should be in a range of 4oC at most so that the Ta would be the same to acquire your fluorescence data alltogether at the same temperature. I think there are some softwares where you can add a second plateau to acquire fluorescence data. So, you could have two Ta; one for the GOI and one for the reference gene.

However in my experience this is not always necessary. I would suggest, if you have time, to run a PCR array once and see if you have bands that correspond to the products you expect. If so, then you are fine (which is quite often the case). If not, you can go on and redesign the primers, as others also said.

I totally disagree with the point made by someone that you can run the housekeeping gene in one plate and the GOI in another. For statistical reasons they have to be in the same plate. What I do when I have many genes is that I run each of them separately in a different plate with its housekeeping gene. Then, using t-test you can compare your results as independent samples.