An improved quality control for bisulfite-PCR-based DNA methylation analysis: cycle threshold value.
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.
Ang
Pei Woon
PW
School of Surgery, University of Western Australia, Nedlands, Australia.
Toh
Hwee Boon
HB
Iacopetta
Barry
B
Soong
Richie
R
D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D019175:DNA Methylation; D016678:Genome / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011786:Quality Control; D015203:Reproducibility of Results; D013053:Spectrophotometry; D013447:Sulfites / Q000378:metabolism*
D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite