Analysis of mRNA from microdissected frozen tissue sections without RNA isolation.
Molecular study of gene expression in solid tumors is based largely on mRNA extracted from crushed frozen tumor samples. As most tumors are heterogeneous in composition, molecular alterations acquired by neoplastic cells may be masked by normal epithelial, stromal, and inflammatory cells, which may make up a significant volume of many tumors. We have developed a technique whereby reverse transcription polymerase chain reaction (RT-PCR) can be performed on lesions microdissected directly from frozen tumor sections. This allows for molecular analysis of mRNA from histologically homogeneous cell populations. Cryostat sections are placed onto a thin layer of 2% agarose on a glass slide and stained briefly. Microdissected tissue is immersed in a freezing solution to lyse the cells; aliquots are used directly in RT-PCR reactions without further purification. We successfully amplified cDNA fragments of the beta2-microglobulin, p21Waf1, and BRCA1 genes from small microdissected lesions. Also, we examined the effect of varying thickness of cryostat sections (20 versus 40 microm) and several tissue staining dyes. We estimate that a small microdissected region, containing no more than 200 cells, can provide enough mRNA to make cDNA for 80 to 100 PCR reactions. We believe that this technique will be a useful tool to study gene expression in histologically defined tissues.
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MD
Department of Molecular and Medical Genetics, University of Toronto, and the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Ontario, Canada.
Done
S J
SJ
Redston
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Andrulis
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IL
D019313:BRCA1 Protein / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D005629:Frozen Sections / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D001613:beta 2-Microglobulin / Q000235:genetics
D019313:BRCA1 Protein; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin