Dear Afshin Seyendnejad,

You should use the EndPoint. Also, check to have it set up in the data collecting software EndPoint as well.

Let me know if you have another questions

KR

Alexei

Dear Dr. Alexei Levitchi

Thanks for your reply.

Would you please explain your answer?

Why choosing Endpoint? What's the difference between Endpoint and real time experiment types?

Dear Afshin Seyednejad,

TaqMan genotyping is a qualitative technique, to determine the expected quality (allele 1, allele 2, ..) in a SNP locus. So, you do not need to know how much DNA/ target sequence copies were at the beginning of the PCR and and at the end. Indifferent to this, you will receive the fluorescence signal of a certain kind.

Regarding the choice - it is normal to use this way as the rule. Besides, there is an important difference in program set up - RealTime needs additional signal measure at the end of each cycle. Otherwise it would not be possible to elaborate graphics.

So, the software needs to know which mode was set up to know

But once you will increase your experience you will not that it does not matter so much, as setting the experiment in RealTime mode, you can easily convert to Endpoint at the stage of results analysis. Also, In TaqMan Genotyper, you can start to analyze same sample set in these two modes separately to compare, but the final interpretation would be very similar (I mean sample set with good quality parameters).

Still, I was applying both these setups at the step when I have to check working flow I was implementing. E.g. how to differentiate true negative result from pcr inhibition in a certain tube - if you would apply EndPoint - you will just see lack of threshold reaching signal and the result would be considered negative, while applying RealTime you can see on the graph that there was some amplification which was very weak OR it was inhibited on a certain step.

Another need appears to validate if a sample is reproducible = whether same sample is generating generally or generating same result in same experiment plate, or batch of plates (a had hundreds of samples check for 20 loci).

So, if you have to analyze small number of samples on the same plate - use the software you collect the data. If you have many samples, or many plates, or many Probes to check than : 1 firstly check each plate results in data collection software, as technical validation step and 2 - go to TaqMan genotyper software.

Besides, you can connect to the ThermoFisher Connect cloud (https://www.thermofisher.com/at/en/home/cloud.html) and use their last versions of (almost) any software.

How it was explicitly.

If you have some more technical or experimental design questions, write me.

Good luck!

Alexei

Dear Dr. Alexei Levitchi,

Thank you so much for your very comprehensive explanation.

I have about 200 samples.

As you mentioned, I have compared my results in these two modes and they were just slightly different. But I wanted to know the reason behind this and also use the most reliable study type.

Thanks again.

Most reliable type is not good phrase, as the meaning are different. For me , it is the case when I can have technically validated and clearly understandable data obtained, and only after this I can have higher (never 100%) trust in the interpretation done. Otherwise, any sample with an unclear interpretation to be repeated. Thus, you will have same interpretation results indifferently which type of set up and data analysis the software will apply.

Did you use repetitions (at least duplications) of your samples? In this way, you should not have doubts on the genotyping results. Otherwise, if you have same sample genotyped differently, you MUST recheck the samples, may be even starting from sample notation, DNA extraction etc.

So, this is what I meant regarding the whole work flow, you must set it up, from DNA extraction until genotype interpretation in the conditions not to accept unclear interpretation.

Good luck!

Yes, I had duplication of my samples and they were consistent and reproducible.

Also, there were no significant differences in results when analyzing in collection software, real time mode and Endpoint mode.

Thanks.