The data obtain from qPCR contains TL/reaction & SCG/reaction. The T/S ratio obtain does not give the expected values. I am not getting the 5-15kb TL values in controls. I am unable to calculate the TL measurement.
I have attach the paper am refering.
First off, the T/S ratio isn't going to be the actual length, merely a relative amount of telomeric DNA per single copy gene, meaning a value of 5 doesn't mean 5 kbp.
If you perform dilution series for both telomere and SCG using some standard (we typically use DNA from normal human fibroblasts for this) you calcualte the relative amount of telomere and SCG DNA (both in ng) compared to your standard. So a sample T/S of 5 would mean you have 5 fold as much telomere DNA than your standard.
Beyond that, verify you are only getting 1 (major) product length by using a melt curve and this PCR seems to lose efficiency without optimized primer concentrations (for us anyway).
Hope this helped some. Good luck
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