In fact, an approach for absolute (rather than relative) telomere length measurement by qPCR has been developed. The following papers provide the protocols in detail:

http://www.ncbi.nlm.nih.gov/pubmed/18476834

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047434/?tool=pubmed

I agree that hybridization techniques are currently considered the gold standards, but they require more DNA than you have (that's why the question was about measuring telomere length using a qPCR-based method). The only disadvantage is that qPCR does not have single chromosome resolution (opposed to FISH on fixed cells, for instance), providing measures concerning mean telomere length per cell only. But normally such a resolution is not of primary interest (at least for the studies I have seen on this topic).

Roche also has the kit for Telomere Length Assay (TeloTAGGG Telomere Length Assay). Or you can follow the papers:

Telomere length measurement by a novel monochrome multiplex quantitative PCR method.

Authors: Richard M Cawthon

Telomere measurement by quantitative PCR.

Richard M. Cawthon

Telomere Length Trajectory and Its Determinants in Persons with Coronary Artery Disease: Longitudinal Findings from the Heart and Soul Study.

Authors: Ramin Farzaneh-Far

Thanks for the references. About Roch kit, it needs 1-2 mirgram DNA which I don't have. I only have couple of hundred nanograms.

Hello Bita,

maybe this paper from 2000 will help you:

http://mcb.asm.org/content/20/5/1659.full.pdf+html

This is working in human and so maybe also possible for your cells (human cells?).

Hello Bita I have been working on telomere as well. I would better suggest you to go for the real-time PCR with the primer being designed by Cawthon. Real-time is better and requires tiny amount of DNA. You can use up to nanogram or picogram of DNA to perform the assay.

qPCR will not determine telomere length in kb but only the abundance of telomeric DNA in your sample.

Golden standards for measuring telomere length are TRF analysis (even though this includes subtelomeric sequence as there are no restriction sites in TTAGGG sequences or telomere FISH with PNA probes via hybrifisation either in situ on fixed cells on a slide or in suspension and analyses by FACS analysis (Flow-FISH).

As Axel has noted, qPCR is only used for determining RELATIVE telomere length, that is relative to a stable reference gene. It depends on your study design, do you want to compare patients and controls, or do you require a precise measure of telomere length? Numerous studies that have compared TL between different groups have used qPCR for relative telomere length determination, it requires small amounts of DNA, is cost effective and is simple and straight forward to perform.

In fact, an approach for absolute (rather than relative) telomere length measurement by qPCR has been developed. The following papers provide the protocols in detail:

http://www.ncbi.nlm.nih.gov/pubmed/18476834

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047434/?tool=pubmed

I agree that hybridization techniques are currently considered the gold standards, but they require more DNA than you have (that's why the question was about measuring telomere length using a qPCR-based method). The only disadvantage is that qPCR does not have single chromosome resolution (opposed to FISH on fixed cells, for instance), providing measures concerning mean telomere length per cell only. But normally such a resolution is not of primary interest (at least for the studies I have seen on this topic).

If you are looking for a telomere phenotype you would want to get as much resolution and information as possible. Is there any chance you can extract more DNA from your cultured cells, Bita?

Agreed (that's why I said it was a disadvantage oh the method). But if resolution is to be pursued that far (at chromosomal level), then I would also work with a number of replicates higher than the usual. It would be more laborious, but equally more informative.

Thanks for all of your suggestions. for the Axel question, I should say , I could culture more cells ,but as I want an exact time point in culture & I need one small sorted population, It is always hard to get more DNA. Anyway as I am going to comare the telomere length of cultured cells to control which is non-cultured cells,I think QPCR might work for it.

Bita, how did you go with the qPCR for Telomere Length? I am keen to know your results. Also could you share your protocol please? Many thanks

pPCR method CAN NOT measure telomere length but just sort of amplification noise signal. I would say most of people using the method may do not understand what exactly occur in the reaction system nor they are interested in knowing that.

Xiong, then which method should be used? Can PCR sequencing work for teleomere lenght/ TTAGGG repeat sequences?

I don't think any sequencing can do telomere length assay.  We have developed a novel technology called Omega-probe mediated RT PCR. 

I followed O'Callaghan and Fenech paper (Biological Procedures Online 2011) and could not repeat their result. The efficiency of Telomere PCR was really bad, and I also found multiple bands in melt curve analysis. I emailed authors and asked for help, but they didn't answer my questions. Do you successfully repeat their qPCR method?

I don't think most of people if not all,  following Cawthon's qPCR understood what's going on during PCR amplification. I have been thinking of his qPCR negatively for telomere length assay but never used the method. We are filling a patent now. The tech Model seems pretty promising.

Hello

There are various methods used to measure telomere length,each with its own advantages and disadvantages for that you must be careful to choos the correct telomere measuring method .

See attachments here will help you

Good Luck 

you can see this article

Thanks Mohammed, that is a very helpful paper.

qPCR provides an easy and accurate way to measure telomere length. You simply needs 2 pairs of primers, one to amplify telomere and the other to amplify a single copy reference on the genome. However, you need to make sure 1) both primer pairs efficiency is close to 100%; 2) no non-specific amplification for both primer pairs, and 3) no primer dimer formation (especially for telomere primer pair) within 30 PCR cycles.

https://www.sciencellonline.com/catalogsearch/result/?q=telomere

https://www.sciencellonline.com/PS/8918.pdf

Daniel,

That is excellent information. Thank you very much!

Tore

Tore,

You are welcome!

Daniel

Comment on Hi Daniel Li post

All use current RT-qPCR tech (Cawthon 2002; 2009) to measure telomere length should very carefully evaluate and understand what really going on in the system during PCR amplifications. One of the simple way to evaluate the assay is: using the same sample, get the Tc value for the telomere and the Tc value for SCG. There should be 1: 10,0000~10,000000 (SCG:telm) per cell, so the Tc value should show different accordingly.

And is telomere lenght measured by those methods a reliable way to study senescence, in normal and pathological models, such as cancer?

Hi Aldo,

Thanks for the recommendation. I wouldn't call telomere length a reliable way, but it's a good indication. In general, senescent cells have shorter telomeres, and most cancer cells have reactivated telomerase activity.

To my best knowledge, since there is no reliable method so far for absolute telomere length assay (TRF somehow shows real telomere length but includes sub-telomeric parts and errors arise from post-data processing) all data regarding to telomere length vs. senescence, cancer and human diseases are contradictory and need re evaluation.

I also just found this paper on TeSLA:

A method for measuring the distribution of the shortest telomeres in cells and tissues

Lai et al. 2017

PMID: 29116081

PMCID: PMC5676791

DOI: 10.1038/s41467-017-01291-z

Has anyone tried this method?

Thanks

Dear Bita,

We are trying to measure absolute Tel length in human leukocytes using O'Callaghan 2011 methods. We have not managed to get a good standard curve from StepOnePlus Real-Time PCR software yet after many efforts. But when we use the log, the standard curve, efficiency and slope are in expected range. any advice ?

Well you can explore our new devloped method it may be of some help.But it is to measure the relative telomere length only

Preprint Novel Dual Labeled Fluorescence Probe Based Assay to Measure...

Hello

There are a lot of methods to measure the length of telomere such as :

Absolute telomere length (aTL), High throughput quantitative fluorescence in situ hybridization (HT Q-FISH) ,Monochrome multiplex quantitative polymerase chain reaction (MMqPCR), Quantitative polymerase chain reaction (qPCR), Single Telomere Length Analysis(STELA), Terminal restriction fragment (TRF), Quantitative fluorescence in situ hybridization(Q-FISH).

Article Telomere Length A Review of Methods for Measurement

Good Luck