In Situ RT Polymerase Chain Reaction (In Situ RT-PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells.
In Situ polymerase chain reaction is a histological technique that exploits the advantages of PCR for detection of DNA or mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for target DNA or mRNA localization in cell organelles, intact cells, or tissue sections. We provide here a detailed protocol for direct in situ reverse transcription (RT), PCR (RT-PCR) and PCR with non-radioactive probes after fixation and paraffin embedding. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with and anti-digoxigenin antibody conjugated with HRP. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling. In our design, this kit could be applied in qualitative analysis and semi-quantitative analysis. The PCR cycles should be optimized by each researcher to the experiment goal.
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. The protocol includes the following steps: (i) tissue preparation, (ii) In Situ PCR (or In Situ RT-PCR), (iii) signal detection. The technique has high sensitivity (geometrically PCR-amplifying 150–350 bp fragments of a gene of interest in situ) and specificity. 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 6 hours is required to carry out the technique.
