DNA content and cell number determination in microdissected samples of breast carcinoma in situ.
Paraffin-embedded tissue (PET) is the specimen of choice for the histopathological diagnosis of cancer. PET has become a valuable resource for correlating cellular phenotype and genotype in microdissected lesions. A definitive improvement in this field has been the development of infra-red laser capture microdissection (LCM), which yields homogeneous populations of cells for DNA extraction and in vitro amplification by polymerase chain reaction (PCR). We report here a photographic and fluorescent technique for determining the number of nuclei and concentration of DNA obtained respectively by laser capture microdissection from paraffin-embedded breast cancer tissue. Breast biopsies containing carcinoma in situ were serially sectioned, mounted on plain glass slides and tumor cells were microdissected using a laser capture microscope. The DNA was extracted in digestion buffer and used directly as a template for PCR. In our protocols, each capture contained 21+/-5.4 nuclei with a DNA concentration of 115+/-5.3 pg. Also, a linear relationship was found between number of captures and DNA content (R2=0.9995). These results represent a novel contribution for a more precise correlation between phenotypic and genotypic diversity in cancer cells studied from microdissected paraffin-embedded tissue.
DeSales University, Center Valley, PA, USA.
Robert
R
Blumenstein
Margarida
M
Dias
Irma H
IH
Russo
Quivo
Q
Tahin
Jose
J
Russo
D001943:Breast Neoplasms / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D002278:Carcinoma in Situ / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D002452:Cell Count; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007834:Lasers; D016610:Tissue Embedding
D004273:DNA, Neoplasm