Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues.
We developed an assay to quantify DNA methylation in breast cancer cells isolated by laser capture microdissection (LCM). The assay uses methylation sensitive restriction enzyme (MSRE) digestion and quantitative polymerase chain reaction (qPCR). To assess the validity and precision of the assay, we prepared standard samples with expected methylation percentage (MP) for two gene promoters (PLAU (plasminogen inhibitor, urokinase) and TIMP3 (TIMP metallopeptidase inhibitor 3)) that we compared with measured MPs. We found good linearity of MSRE digestion and qPCR procedures for both promoters (beta=0.90-1.19+/-0.05-0.10 and r=0.95-0.98; all P<0.0001). Moreover, results remained similar after addition of a purification step between MSRE digestion and qPCR procedures. The validity of this technique was also confirmed by successfully replicating previously published MPs of four cell lines for PLAU and TIMP3 promoters. We assessed the consistency of our approach by comparing MPs of PLAU and TIMP3 promoters from nine breast cancer patients and two cell lines using LCM frozen tissues and their corresponding formalin-fixed paraffin-embedded tissues. We found good consistency (intraclass correlation coefficient=0.93) of MPs between frozen tissues and formalin-fixed paraffin-embedded tissues. Our data demonstrate that this assay based on digestion with MSRE and qPCR procedures is a good technique to quantify MP on limited amounts of DNA and may find clinical applications.
Gagnon
Jean-François
JF
Service de pathologie, Centre hospitalier affilié universitaire de Québec, Québec, Canada.
Sanschagrin
François
F
Jacob
Simon
S
Tremblay
Andrée-Anne
AA
Provencher
Louise
L
Robert
Jean
J
Morin
Carol
C
Diorio
Caroline
C
D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D005557:Formaldehyde; D020868:Gene Silencing; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D016707:Tissue Fixation; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; D055293:Receptors, Urokinase Plasminogen Activator; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D005557:Formaldehyde; D004262:DNA Restriction Enzymes