title: An improved quality control for bisulfite-PCR-based DNA methylation analysis: cycle threshold value.
abstract: 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.
authors: School of Surgery, University of Western Australia, Nedlands, Australia.
authors: Pei Woon
authors: PW
authors: Ang
authors: Hwee Boon
authors: HB
authors: Toh
authors: Barry
authors: B
authors: Iacopetta
authors: Richie
authors: R
authors: Soong
mesh_terms: 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*
chemical_list: D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite