PCR-in situ for Histopatology
PCR-in situ for Histopatology
The use of in situ RT-PCR to examine gene expression in disease tissues has certain advantages over more established hybridisation, PCR amplification or antibody-based techniques. As with immunohistochemistry, detection of gene expression is at the level of individual cells, but whereas polyclonal antibody production by immunisation may take 4 months or longer, and require extensive optimisation, it is relatively easy to characterise and optimise oligonucleotide primers which have considerably less chemical complexity and therefore, inherently more predictable properties. Moreover, while cross-reactivity is a frequent problem when selecting antibodies for protein detection, it is a simple matter to select PCR primers that are specific to a single member of a gene family, or even a particular splice variant of that gene (Heid, 1996).
We have successfully applied in situ RT-PCR to 1 mm paraffin-embedded tissue section arrays in order to determine which cells within a cancer are responsible for gene over-expression observed in RNA extracts. A number of technical manipulations were incorporated into the in situ protocol to ensure specificity and fidelity, and these transferred readily to the micro-array format. To our knowledge, this is the first time this procedure has been applied simultaneously to multiple samples in a microarray format.
A DNAse digestion step is commonly used in RT-PCR amplification in order to reduce the risk of spurious amplification of genomic DNA. This can also be carried out on tissue sections but the extensive incubation time required (up to 16 hr) means that considerable tissue autolysis occurs, damaging tissue structure and making post-PCR identification of cells difficult. In our protocol, the DNAse digestion step was omitted so as to better preserve tissue structure. Modifications to experimental design were employed to prevent amplification of genomic DNA. Although other approaches have been taken to obviate nuclease pre-treatment, we employed more conventional means. Firstly, primers were designed to amplify across two different exons, so that the amplified fragment from reverse transcribed, fully spliced mRNA would be small (300 bp), whilst the distance between the same primer sites in genomic DNA was over 3500 bp. Secondly, the number of PCR cycles and the duration of the polymerisation step were minimised so that any priming from genomic DNA would fail to achieve chain-reaction amplification. These strategies had a number of other beneficial effects: the PCR cycle number was kept with the linear range of amplification established by real-time quantitative RT-PCR, giving a more quantitative representation of the mRNA remaining in each cell, and avoiding significant synthesis of non-specific artifacts. Diffusion of reaction products away from the site of synthesis, another problem associated with in situ PCR, was reduced by this rapid procedure and exposure of the tissue sections to destructive conditions was also minimised, with the result that post-amplification staining revealed a high degree of preservation of tissue architecture and cellular features.
A consequence of using low PCR cycle numbers is that the degree of amplification will be limited, with implications for detection of the PCR product. Standard peroxidase-linked antibody detection is insufficiently sensitive. Chemiluminescent or fluorescent detection reagents could be used instead to amplify the signal, but these would require specialised image detection systems and would rapidly diffuse away from the point of detection. Immunogold labelling followed by silver nucleation produced solid particles visible by light microscopy at magnifications suitable for visualising tissue and cellular features. This enabled simultaneous imaging of PCR products and hematoxylin-stained tissue details. The silver particles were bound to PCR products via anti-digoxygenin antibodies, and proved resistant to diffusion, remaining in the same cellular localisation as the original mRNA.
A persistent problem with the in situ PCR procedure has been inconsistency of results. Dedicated instrumentation has been designed with the aim of controlling conditions on a microscope slide, and some machines accommodate four or more slides to increase throughput and lower experimental variability. However, variation in the quality of paraffin-embedded tissue sections, and the number of steps involved in in situ PCR and the time taken to acquire data on significant numbers of samples affect the reproducibility of the technique. We found that a single, standard in situ PCR coverslip covered up to seventy 1 mm sections on a Clinomics tissue microarray, enabling simultaneous amplification of reverse transcribed RNA in each section under selected conditions. Although small tissue sections are more likely to become dislodged during the process of de-waxing and amplification, the use of poly-L-lysine coated slides decreased these losses and the cancer tissues examined were intrinsically more adherent due to their high cellularity. Thus a significant number of tissue samples could be analysed per single experiment. This approach substantially addresses the problem of slide-to-slide variability by subjecting large numbers of samples to identical experimental conditions. In addition, our technical modifications minimised tissue damage during preparation and amplification, preserving useful information on cellular morphology (Staecker, 1994).
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