Reverse Transcriptase PCR In Situ on Cryopreserved Tissue Sections

The polymerase chain reaction (PCR) is an extraordinarily powerful tool that can amplify fragments of DNA or mRNA from a single cell (1 ). By combining PCR with other established methods, the reaction has been extended from isolated DNA in solution, to mRNA, to studies using tissue sections (2 –5 ). In situ PCR is much more sensitive than standard in situ hybridization (ISH) because the cellular signal is amplified (6 ). It has been used to detect unique intracellular nucleic acid sequences (5 ). Most investigators have amplified the DNA or RNA on the tissue section and used standard ISH to identify the amplified product (PCR-ISH) (2 –4 , 6 ). In PCR in situ , the solution amplification has been adapted to tissue sections, and the segment is amplified directly and demonstrated using antibodies or ligands to modified, incorporated nucleic acids or using autoradiography. The assay requires oligonucleotide primers that are specific for the DNA/mRNA segment of interest. Nucleotides modified with digoxigenin, biotin, or radioactivity are incorporated into the amplified product that allows detection of the amplified segment avoiding potential nonspecific binding associated with ISH. We have developed the assay so that it can be used on tissues adherent to slide fragments that are cut to fit into 500-�L thin-walled PCR tubes (7 ) and to full-size slides (8 ). The use of slide fragments allows the assay to be done under conditions that are identical to standard PCR with small, 3 � 3 mm, pieces of tissue. The assay on whole slides permits the use of tissue sections 1 cm in diameter.