NONRADIOACTIVE LOCALIZATION OF NUCLEIC-ACIDS BY DIRECT IN-SITU PCR AND IN-SITU RT-PCR IN PARAFFIN-EMBEDDED SECTIONS

Technological developments have made possible extension of polymerase chain reaction (PCR) analysis to individual cells to localize DNA/RNA with non-radioactive labels at the light microscopic level, This appro ach, in situ PCR, is particularly useful in resolving low-frequency me ssage expression in mixed populations of cells and tissues, We have es tablished a working protocol for direct in situ PCR and have utilized several controls to validate our results, In this report we outline th e procedures for detecting either DNA or RNA in a rapid and reproducib le manner, We evaluate the sequential steps required for this analysis , such as protease hydrolysis, DNAse digestion, ''hot start'' capabili ties, and detection methods, We have applied these methods in several applications, including detection of the p53 gene in human tumor sampl es, localization of insulin-like growth factor-IA mRNA in cell lines w ith low levels of expression, and distribution of transferrin mRNA in lung cancer cell lines and tumors, We demonstrate from this study that the in situ PCR technique is an investigative approach capable of det ecting specific DNA/RNA sequences at the cellular level and of identif ying cells with low levels of mRNA expression.