In Situ PCR
Definition of In Situ PCR
In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells.
In Situ PCR Introduction
During the initiation and progression of disease, minute quantities of a product in small populations of cells or tissues may be vital for the pathogenesis of the disease.
In many slowly-evolving diseases which require months or even years to manifest themselves clinically, it has been shown that the majority of the affected cell population is in a transcriptionally inactive state, and at a level of one gene per host cell.
Nucleic acid hybridization methods and the polymerase chain reaction (PCR) have both been employed to examine the expression and detection of such affected genes during pathogenesis. While both these techniques are quite useful, the disadvantage of these techniques is that they are essentially conducting cell expression and population studies. Nucleic acids are isolated from a population of cells which contains either a sufficient number of molecules to detect directly by standard hybridization techniques, or, when a subpopulation contains as little as a single copy of nucleic acid, that molecule amplified by the PCR, and detected after amplification.
In situ hybridization (ISH) applies the methodology of the nucleic acid hybridization technique to the cellular level. Combining cytochemistry and immunocytochemistry, In Situ PCR allows the identification of cellular markers to be identified, and further permits the localization of to cell specific sequences within cell populations, such as tissues and blood samples.
In Situ PCR is limited to the detection of non-genomic material such as RNA, genes or genomes, as the detection limit in most conditions is several copies of the target nucleic acid per cell. Therefore, due to copy number limitations, hybridization of RNA is more sensitive than DNA detection.
Factors affecting In Situ PCR sensitivity include:
1) the strandedness of the target molecule
2) the lack of a complementary sequence proximal to the target sequences
Reverse transcriptase-catalyzed in situ transcription has even been used to detect RNAs which occur at relatively high copy number.
In Situ PCR (ISH) Protcol
Initially the stoichiometry of the nucleic acid components makes the reaction primer-driven. As the reaction proceeds however, the PCR product itself begins to function as a target for further amplification, as well as a primer for further elongation. It is the accumulation of sequences elongated in multiple reactions which is retained by the fixed cell and acts as the target for the radiolabeled probe in the detection phase.
The complex physical nature within a fixed cell impedes both the efficiency of the polymerase as well as diffusion and annealing, and thus elongation times are much longer than those conducted under aqueous (standard PCR) conditions. Optimum signal is usually producted using a 15 minute elongation.