Protocols for the in situ PCR-amplification and detection of mRNA and DNA sequences

Abstract

In this protocol we describe the in situ PCR method for the amplification of both DNA and mRNA targets [in situ reverse transcriptase-PCR (RT-PCR)], from frozen or paraffin-fixed tissue sections, cell culture or other single-cell suspensions. Detection of amplicons can be achieved by the hybridization and detection of labeled probes. The protocol includes the following steps: (i) tissue preparation, (ii) in situ PCR (or in situ RT-PCR), (iii) probe hybridization, (iv) signal detection. The technique has high sensitivity (geometrically PCR-amplifying 150-350 bp fragments of a gene of interest in situ) and specificity (derived from in situ hybridization with specific fluorescent or biotinylated probes for the target genes). The ability to identify individual cells, expressing or carrying specific genes of interest in a latent form in a tissue section under the microscope provides a visual account of silent genes, and allows the determination of various aspects of normal versus pathological conditions, or latent versus active viral replication. An average of 48 h is required to carry out the technique.