Hi Kailash, 

The traditional method of measuring telomere length in samples of total genomic DNA determines a mean terminal restriction fragment (TRF) length (1). The method requires large amounts of DNA (0.5–5 µg/individual) and time (3–5 days). Furthermore, the relative mean TRF lengths of individuals can vary by as much as 5% depending on the particular restriction enzymes used, suggesting the existence of subtelomeric restriction site polymorphisms and/or subtelomeric length polymorphisms that may confound the identification of primary factors accounting for inter-individual variation in the mean length of the true telomeric repeat sequence. More recently, methods have been developed that allow multiple samples to be compared for their relative content of just the telomeric hexamer repeat itself (2–5). However, none of these methods is as simple and as amenable to rapid high throughput processing of large numbers of samples as the method presented below.

Our strategy for determining relative telomere lengths by quantitative PCR was to measure, for each DNA sample, the factor by which the sample differed from a reference DNA sample in its ratio of telomere repeat copy number to single copy gene copy number. This ratio should be proportional to the average telomere length. The quantity of telomere repeats in each experimental sample was measured as the level of dilution of an arbitrarily chosen reference DNA sample that would make the experimental and reference samples equivalent with regard to the number of cycles of PCR needed to generate a given amount of telomere PCR product during the exponential phase of PCR amplification. Similarly, the relative quantity of the single copy gene in each experimental sample was expressed as the level of dilution of the reference DNA sample needed to match it to the experimental sample with regard to the number of cycles of PCR needed to generate a given amount of single copy gene PCR product during the exponential phase of the PCR. For each experimental sample the ratio of these dilution factors is the relative telomere to single copy gene (T/S) ratio. Thus T/S = 1 when the unknown DNA is identical to the reference DNA in its ratio of telomere repeat copy number to single copy gene copy number. The reference DNA sample (to which all of the experimental samples in a given study are compared) can be from a single individual or it can be a pooled sample from multiple individuals. The T/S ratio of one individual relative to the T/S ratio of another should correspond to the relative telomere lengths of their DNA.

Here are some of the links which will help to you..

http://www.ncbi.nlm.nih.gov/pubmed/21369534

http://www.biotechniques.com/BiotechniquesJournal/2008/May/A-quantitative-real-time-PCR-method-for-absolute-telomere-length/biotechniques-45204.html

http://www.jove.com/video/50246/telomere-length-telomerase-activity-yin-yang-cell

http://www.legalmedicinejournal.com/article/S1344-6223(08)00027-8/abstract?cc=y

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC115301/

Thank you Prudhvi for you suggestion and explanation of technique. My major concern is about how to decide the primer set for telomere amplification, since two kind of primers are mentioned in literature?

Follow the link

Estimating telomere length

http://nar.oxfordjournals.org/content/30/10/e47.full

Estimating telomere length from whole genome sequence data

http://nar.oxfordjournals.org/content/early/2014/03/07/nar.gku181.long

Thank you Salwa for your suggestion.

I am using conventional TRF method with HinfI and RsaI enzymes for my work. As prudhvi stated, TRF analysis is laborious, time consuming protocol, need more quantity of DNA etc., 

Here I have attached JOVE video protocol link... and .pdf file as well. 

Hope this would help you!

Ramanan Devaraj

http://www.jove.com/video/50246/telomere-length-telomerase-activity-yin-yang-cell

Prudhvi Chand Mallepaddi

Prodhvi's recommendation is absolutely wrong. It seems people have to really get into what happen during RT-qPCR. I was supervised the telomere absolute length measurement paper could get published. The author seemed not really understood RT-qPCR.

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

Hi I am doing work on telomere and optimizing temperature by simple conventional pcr by tel primers as mentioned in cawthoan...but getting dimers.had changed temperatures , concentrations but no progress....each time same result

Cawthon's qPCR methods (2002, 2009) have sort of inherent defects. While a typical qPCR runs a ~100 bp fragment amplification along a much much larger/longer target using a pair of special primers, thinking about qPCR along a telomere target, many primers bind along a telomere one after another, running highly heterogeneous qPCR reactions which do not follow or even completely broken the principle of qPCR quantification.

I am a high school teacher and one of my students wants to do telomere length for fruit flies. I have a thermocycler but not a qPCR. How do I find a protocol for Real-time PCR but not qPCR?

As far as I know, there is no PCR protocol which really works for whole telomere length assay.

BTW, I'm not sure what you mean "real-time PCR but not qPCR". To my best knowledge, most of time, they both refer to the same or similar issue, i.e. quantify the copy number of a target using a PCR-base assay.

there are currently PCR-base protocols for telomere length (relative) assays. you can search by "Cawthon, 2002; 2009" although as I mentioned before the protocols are wrong in term of precise quantification of telomere length.

Dear Dr. Xiong,

we are thanks to you for explaining why you are not sure about Cawthon methods?

Thank you

Hello

You can try with one of these methods:

1-Monochrome multiplex quantitative polymerase chain reaction (MMqPCR)

Article Cawthon RMTelomere length measurement by a novel monochrome ...

2-Quantitative polymerase chain reaction (qPCR).

Article Telomere measurement by quantitative PCR

Good luck

Dear Dr. Saleh Alkarim,

Thank you for your advice.