A hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) method for genomic DNA methylation assessment by UV detection.
A hydrophilic interaction chromatography-based method, in combination with 1.7 microm ethylene bridged hybrid particle packed column (100 mm x 2.1 mm I.D.) and ultraperformance liquid chromatography, has been developed to measure cytosine (C) and methylcytosine (mC) in order to evaluate the extent of DNA methylation. Separation of cytosine and methylcytosine was achieved with good resolution and in fairly short times (5.5 min) by using isocratic elution with a mixture of 97:3 (v/v) acetonitrile/10 mM ammonium acetate as a mobile phase. The determination coefficients of C and mC were high (R(2) > 0.999) within the range tested. The %RSD for intraday and interday were respectively 2.2% and 2.5% for C and 3.5% and 3.8% for mC. The limit of detection was 0.52 microM (0.52 fmol on-column) both for C and mC while the limit of quantification was 1.72 microM (1.72 fmol on-column) both for C and mC. The smallest amount of purified DNA that yielded a measurable level of C and mC was 10 microg. On the whole, this method is simple, rapid, sensitive, and precise.
Sotgia
Salvatore
S
Department of Biomedical Sciences and Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, 07100 Sassari, Italy. salvatore.sotgia@gmail.com
Zinellu
Angelo
A
Pisanu
Elisabetta
E
Murgia
Luciano
L
Pinna
Gerard Aime
GA
Gaspa
Leonardo
L
Deiana
Luca
L
Carru
Ciriaco
C
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis; D019175:DNA Methylation; D015894:Genome, Human; D006801:Humans; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014867:Water / Q000737:chemistry
D014867:Water; D004247:DNA; C427203:calf thymus DNA