title: Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.
abstract: Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China.
authors.forename: Zhaojun
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China.
authors.forename: Liankun
authors.initials: L
authors.lastname: Gu
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China.
authors.forename: Dajun
authors.initials: D
authors.lastname: Deng
mesh_terms: D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D024043:Collagen Type II / Q000032:analysis / Q000302:isolation & purification; D018899:CpG Islands / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019175:DNA Methylation / Q000235:genetics*; D005753:Gastric Mucosa / Q000473:pathology; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000032:analysis / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology
chemical_list: C000614131:CDKN2A protein, human; C499177:COL2A1 protein, human; D024043:Collagen Type II; D019941:Cyclin-Dependent Kinase Inhibitor p16; C494987:GFRA1 protein, human; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D004247:DNA
keywords: DHPLC; DNA methylation; MSP; MethyLight; false negative detection