Analysis of DNA methylation of multiple genes in microdissected cells from formalin-fixed and paraffin-embedded tissues.
A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.
Dietrich
Dimo
D
Epigenomics AG, Kleine Präsidentenstr. 1, 10178 Berlin, Germany. dimo.dietrich@epigenomics.com
Lesche
Ralf
R
Tetzner
Reimo
R
Krispin
Manuel
M
Dietrich
Jörn
J
Haedicke
Wolfgang
W
Schuster
Matthias
M
Kristiansen
Glen
G
D000273:Adipose Tissue / Q000737:chemistry / Q000473:pathology; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000737:chemistry / Q000473:pathology; D019175:DNA Methylation; D005260:Female; D005348:Fibrocystic Breast Disease / Q000737:chemistry / Q000473:pathology; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007202:Indicators and Reagents; D007834:Lasers; D016246:Lymphocytes, Tumor-Infiltrating / Q000737:chemistry; D042282:Microdissection; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D017154:Stromal Cells / Q000737:chemistry; D013447:Sulfites
D014408:Biomarkers, Tumor; D005404:Fixatives; D007202:Indicators and Reagents; D013447:Sulfites; D005557:Formaldehyde; C042345:hydrogen sulfite