Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection
Laser-assisted microdissection (LAM) allows isolation of specific cell populations for molecular studies. The combination of LAM and of real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) enables generation of quantitative cell-specific gene expression data. Histochemical stains used to identify cells desired for LAM should provide acceptable morphology and not interfere with RNA or with subsequent molecular analysis techniques. To determine a reliable stain for analysing RNA, using the housekeeping gene, RPL13A, we performed quantitative gene expression analysis of laser microdissected cells from prostatic frozen tissues. The frozen sections were histochemically stained with hematoxylin, methyl green, toluidine blue O and May-Grunwald. After laser microdissection real-time quantitative RT-PCR was performed. Methyl green yielded more RT-PCR product than did the other dyes. The lowest yield of amplification was obtained after May-Grunwald staining. Therefore we recommend methyl green for general use in gene expression analysis, especially when handling small amounts of RNA.
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Document Type: Research Article
Affiliations: Institute of Pathology, University of Bonn, 53011 Bonn, Germany
Publication date: January 1, 2003
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- The International Journal of Molecular Medicine is a monthly, peer-reviewed journal devoted to the publication of high quality studies related to the molecular mechanisms of human disease. The journal welcomes research on all aspects of molecular and clinical research, ranging from biochemistry to immunology, pathology, genetics, human genomics, microbiology, molecular pathogenesis, molecular cardiology, molecular surgery and molecular psychology.
The International Journal of Molecular Medicine aims to provide an insight for researchers within the community in regard to developing molecular tools and identifying molecular targets for the diagnosis and treatment of a diverse number of human diseases.
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