I have been working on measuring absolute telomere length from genomic DNA isolated from cultured cells using Callaghan's (Tel/36B4) qPCR method and it has worked quite good. Now I want to measure the absolute telomere length in cell-free DNA isolated from serum, can I use the same protocol? usually cell free DNA are fragmented and hence the reference gene 36B4 won't be suitable. There is another protocol which makes use of LINES as reference sequence and it is published in oncotarget. But before jumping on cloning LINES for generating standard, i want to know whether Callaghan's method will help?
Hello
Yes , you can use quantitative PCR
Look at references here
Good Luck
http://www.nature.com/articles/srep38084
https://www.openaire.eu/search/publication?articleId=od_______908::1a8ccfefdd6761e865e59259497f9f32
I have already read both the paper. The first paper have utilized the whole blood for measuring telomere length and the second paper have used LINES on cfDNA which i have already talked about in my question.
I have finally found the paper, will give a try and soon gonna update about the results.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324593/
If anyone is planning for telomere length measurement from cell free DNA using Cawthon method, go for it. But make sure you have atleast 400 ul of serum sample (stored at -80 C) and elute the cell free DNA in 30 ul of elution buffer. While performing the PCR, use atleast 2 ul of template DNA in 10 ul of reaction. Use the extracted DNA as quick as you can as the yield of DNA is always low in the first place and while freezing, the DNA concentration reduces furthermore. I am getting excellent amplification peaks. If you want to perform the absolute measurement using Callaghan method, use LINES primers instead of 36B4 (make sure you have plasmid DNA containing the LINES sequences for standard curve though).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology