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Problem with Telomeric Length measurement using RT-PCR - (Sep/17/2010 )

Need an advice!

I'm looking at Telomeric length using Telomeric primers developed by cawthorn with some modification by ME Gil and Coetzer, 2004.

I have optimized the assay of Telomere primers and single gene copy 36B4 using Roche Lightcycler 480. I have done validation curve for both the assay and the Regression curve were both more that 0.99. However, the problem lies onto the efficiency of Telomere. For Telomere, I have repeated 3 times with R2 for all of 3 were >0.99, however I got different efficiency such as 2.2, 2.1. and 1.75. For 36B4, The efficiency lies between 1.7-1.9.

I have also tried on the samples, however, everytime I'm repeating the same sample, it give me different value. The value expressed in delta Ct.

I just wonder whether it is possible to get different results due to amplifying repetitive sequence such as Telomere? If not, how this possible? I mean whether it amplyfing primer dimer or unspecific products? can I still use the primers eventhough it give me 2 different value in the same sample?




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