Background: The diagnostic potential of DNA methylation has generated a need for quality control systems in its analysis. Here, we tested whether cycle threshold (Ct) values from real-time quantitative PCR correlate better with the reliability of bisulfite-PCR methylation analyses than spectrophotometric concentrations.
Methods: A total of 18 cell line DNA samples were quantified for beta-actin (ACTB) Ct and spectrophotometric values from 1-microL volumes. A further 1 microL was analyzed in quintuplicate for bisulfite DNA ACTB by MethyLight. Correlations between Ct and spectrophotometric values, and MethyLight qualitative (detection in all replicates) and quantitative (Ct value standard deviation <1) reliability were compared. To validate the findings, the same comparisons were made in 40 formalin-fixed, paraffin-embedded tissue (FFPET) samples analyzed by methylation-specific PCR of MLH1.
Results: Using optimal thresholds, Ct values correctly identified 6/7 (86%) and 11/11 (100%) cell line samples to be qualitatively, and 7/7 (100%) and 4/4 (100%) quantitatively reliable and unreliable, respectively. The corresponding frequencies for spectrophotometry were 6/8 (75%), 10/10 (100%), 6/8 (75%) and 2/3 (67%), respectively. In FFPET DNA, the respective values for qualitative reliability for Ct values were 25/25 (100%) and 11/15 (73%), and 11/14 (79%) and 8/26 (31%) for spectrophotometry.
Conclusions: Ct values could be useful indicators for gauging the suitability of samples for methylation analysis, in particular from FFPET.