Are you doing these experiments to comparte two groups (say, disease cases and controls)? If so, you could try an approach to measure the relative telomere length (as described in the attached PDF), because you would be interested in the differences between the groups, not in the absolute telomere length per say.

Just an extra comment: some recently published high-quality papers I have seen have used the relative measurement approach, perhaps because of its simplicity and for being as informative as the absolute approach when it comes to compare two or more groups (of course it is less informative regarding the telomere length itself, but having this value is not the goal of the experiment in several cases).

Dear Fernando,

Thank you so much for your kind answer and your generous support. Now, the first thing is that, yes, I am working to compare the length of telomere in three different groups in my experiment: control, diabetes and diabtes complicated with CAD subjects. Since I am following the above paper which give us the absolute telomere length, I think that would be okay too.

When I calculate my T/S ratio it is something like in ..... *10^5 kb which can't be possible. I don't know where I am doing wrong in calculation.

No problems, I'm glad I can be helpful :-)

I agree that the paper that the methodology that informs the absolute mean telomere length (I think it's important to keep in mind that quantitative real-time PCR is not sensitive regarding interchromosomal telomere length variations in a single cell level, which are known to occur and to be biologically relevant) should be OK. Unfortunately, I have been working only with the relative approach, so I would need to read the paper you are refering too in order to get a better sense of the methodology we are discussing. I'll go through the paper (thanks for sharing it by the way) and will post here any considerations that might be helpful to your experiments. Nevertheless, I think that one thing we should consider when choosing between two methodologies (in this case, the relative vs the absolute) is to look for others' publications. Based on the literature I have been reading, the relative approach has been preferentially used. Surely it does not mean that the absolute method is not reliable, but it might indicate that people have been finding problems with such method and are then using the relative approach. I mean, considering the absolute approach is more informative than the relative one (and things like cost and time are similar if not the same), why wouldn't people choose the absolute approach? One possible answer is that they did, but found issues that did not occur when using the alternative method.

In case I have any other insight after reading the paper, I'll let you know. Best of luck with your experiments!!

Well, I just finished reading the paper. The strategy is indeed interesting. Before pointing out some insights, the answer to your question in the first post about the value you get is that, since you are dividing by 92, the final result is the mean telomere length / genome, considering both ends of the chromosomes.

Bellow there are some points I though are more likely to affect the results:

- The very first and obvious concern is about the efficiency of the primers. Although they seem to be nice in the paper, inter-lab variation is a common issue. If your SCG primer pair is resulting in poor amplification, for example, then it could result in very large aTL values.

- Another very obvious consideration concerns not analyzing the results that are not contained in the linear range (and considerations regarding standard deviation).

- To normalyze for the number of genomes, it is necessary to remeber to divide the number of copies of the SCG by 2, since it has two copies in the normal human genome (diploid). Then, this value can be used to normalize the values obtained for T. OBS: That's why you divide the T/SCG ratio by 92 to get the mean telomere length; if you would not divide the SCG value by 2 before calculating T/SCG, you would divide this rate by 46 only... It would be somewhat ilogical since the genome is diploid, but it does make sense mathematically.

Again, both the T and the SCG are obtained from the Ct, based on the standard curve. So, having a well-defined and reproducible standard curve seems to be crucial. If I had to guess what is happening in your calculations, I would say that something might be happening when converting the Ct values into either kb (for T) or diploid genomes (for SCG), but this is just a crude guess.

Attached there is a paper published by the same group, also about measuring aTL using qPCR. I thought it could be usefull to you in someway.

Hope this is of any value. Again, best of luck. In case you want to extend the discussion, feel free to do so. It would be fun! :-)

Thank you so much, Fernando.

I am following your suggestions. Thanks a lot.

Lets see.

Have a great time ahead.

I will be writing you shortly.

Dear Fernando,

I have been able to figure it out. Thank you so much for all your kind assistance. It was just problem with the calculation and conversion. :-)

Thank you so much.

Have a great time ahead.

See you around.

With warm regards,

Rajesh

Hi,

Cool, I'm glad you could figure the problem out!

Well, it seems I'm good at guessing!! It was a pleasure to be helpful :-)

You're welcome. The discussion was really fun!

Have a great time too. Best of luck with further experiments.

Best wishes!!

Hii rajesh,

The actual length of human telomeres is approximately 8-15kilo bases.because when a cell divides its telomeres become shorter and shorter as number of cell division increases.

When it becomes too short, the cell can no longer divide, and activate DNA damage response to arrest cell growth and enter Senescence stage or apoptosis.

Thank you Harikrishna and Aria for your kind comments. I solved it successfully. Thank you so much.

Im also controlling telomere from patients of different ages but sometimes the Kb is well over 20 kb do you think I have divide the number for 46 or 23?