ANALYSIS OF MESSENGER-RNA 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 neopla stic cells may be masked by normal epithelial, stromal, and inflammato ry cells, which may make up a significant volume of many tumors. We ha ve developed a technique whereby reverse transcription polymerase chai n reaction (RT-PCR) can be performed on lesions microdissected directl y from frozen tumor sections. This allows for molecular analysis of mR NA from histologically homogeneous cell populations. Cryostat sections are placed onto a thin layer of 2% agarose on a glass slide and stain ed briefly. Microdissected tissue is immersed in a freezing solution t o lyse the cells; aliquots are used directly in RT-PCR reactions witho ut further purification. We successfully amplified cDNA fragments of t he beta(2)-microglobulin, p21(Waf1), and BRCA1 genes from small microd issected lesions. Also, we examined the effect of varying thickness of cryostat sections (20 versus 40 I.cm) and several tissue staining dye s. 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 st udy gene expression in histologically defined tissues.