Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays.
Detection of disease-associated gene transcripts in primary disease tissues is frequently confounded by the presence of non-involved cell types. Alternative methods of detecting gene expression directly within tissues involve either the generation of antibodies, which can be a lengthy process and may suffer from lack of specificity, or amplification of reverse-transcribed cDNA in tissue sections (in situ RT-PCR). The latter method is highly specific and enables detection of transcripts in the cells originally responsible for their synthesis, but is highly destructive of tissue structures and can be carried out on only one or a few sections per experiment, resulting in low reproducibility. In this study, in situ RT-PCR was applied for the first time to commercially available tissue section microarrays enabling the examination of up to 70 different samples simultaneously. Modifications to the technique are detailed that preserved visible tissue and cellular structures and improved transcript detection whilst preventing significant generation of artefacts.
Stamps
Alasdair C
AC
Oxford GlycoSciences (UK) Ltd, The Form, 86 Milton Park, Abingdon, UK OX14 4RY. astamps@ntlworld.com
Terrett
Jonathan A
JA
Adam
Paul J
PJ