TY - JOUR
T1 - An improved quality control for bisulfite-PCR-based DNA methylation analysis : cycle threshold value
AU - Ang, P.W.
AU - Toh, H.B.
AU - Iacopetta, Barry
AU - Soong, R.
PY - 2008
Y1 - 2008
N2 - 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-mu L volumes. A further 1 mu L 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.
AB - 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-mu L volumes. A further 1 mu L 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.
U2 - 10.1515/CCLM.2008.217
DO - 10.1515/CCLM.2008.217
M3 - Article
C2 - 18724808
SN - 1434-6621
VL - 46
SP - 1117
EP - 1121
JO - Clinical Chemistry and Laboratory Medicine
JF - Clinical Chemistry and Laboratory Medicine
IS - 8
ER -