Expression profiling of mouse endometrial cancers microdissected from ethanol-fixed, paraffin-embedded tissues.
Expression-profiling studies have helped define genetic changes associated with carcinogenesis. Determining which alterations in gene expression are causally associated with cancer and which result from the general dysregulation in gene expression that is characteristic of malignancies remains a problem. Transcriptional profiling of early lesions (small cancers or precancers) holds promise for identifying biologically important changes in gene expression. There are, however, technical barriers to the study of small tumors. The total number of cells available for analysis is limiting. It is also often difficult to distinguish cancer cells from normal proliferating cells in frozen sections that are typically used as a source of RNA. Here we describe an ethanol fixation and paraffin-embedding protocol that preserves tissue architecture and cellular morphology of the mouse endometrium, and allows for the recovery of high-quality RNA from microdissected cells. We performed GeneChip expression profiling using RNA from 800 to 4400 cells microdissected from ethanol-fixed, paraffin-embedded uteri. Endometrial adenocarcinomas exhibited changes in the levels of a number of messages known to be abnormally expressed in cancer, and differential expression of additional transcripts not previously implicated in carcinogenesis. We confirmed increased Amd1 expression in RNAs from mouse endometrial carcinomas that were hybridized to GeneChips and validated overexpression of this transcript in additional tumors.
Kabbarah
Omar
O
Division of Biology and Biomedical Sciences and the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63106, USA.
Pinto
Karen
K
Mutch
David G
DG
Goodfellow
Paul J
PJ
D000818:Animals; D019921:Biotinylation; D016172:DNA Fingerprinting; D016889:Endometrial Neoplasms / Q000235:genetics* / Q000473:pathology*; D000431:Ethanol; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D012334:RNA, Neoplasm; D000431:Ethanol