Non-radioactive 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 approach, in situ PCR, is particularly useful in resolving low-frequency message expression in mixed populations of cells and tissues. We have established a working protocol for direct in situ PCR and have utilized several controls to validate our results. In this report we outline the procedures for detecting either DNA or RNA in a rapid and reproducible manner. We evaluate the sequential steps required for this analysis, such as protease hydrolysis, DNAse digestion, "hot start" capabilities, and detection methods. We have applied these methods in several applications, including detection of the p53 gene in human tumor samples, localization of insulin-like growth factor-IA mRNA in cell lines with 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 detecting specific DNA/RNA sequences at the cellular level and of identifying cells with low levels of mRNA expression.
Martínez
A
A
Biomarkers and Prevention Research Branch, National Cancer Institute, National Institutes of Health, Rockville, MD 20850-3300, USA.
Miller
M J
MJ
Quinn
K
K
Unsworth
E J
EJ
Ebina
M
M
Cuttitta
F
F
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D003851:Deoxyribonucleases / Q000378:metabolism; D019286:Endopeptidase K; D005557:Formaldehyde / Q000737:chemistry; D016158:Genes, p53; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D012697:Serine Endopeptidases / Q000378:metabolism; D014158:Transcription, Genetic; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011108:Polymers; D012333:RNA, Messenger; D014168:Transferrin; D005557:Formaldehyde; D012313:RNA; D007334:Insulin-Like Growth Factor I; D004247:DNA; D003851:Deoxyribonucleases; D012697:Serine Endopeptidases; D019286:Endopeptidase K; C003043:paraform