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Real-time quantitative RT–PCR after laser-assisted cell picking

Nature Medicine volume 4, pages 1329–1333 (1998)Cite this article

An investigation of cell type-specific gene expression in intact tissues and an understanding of its regulation requires mRNA quantitation in minute numbers of cells. For this purpose, the sensitivity of PCR has to be complemented by the precision achieved with northern blotting or RNase-protection assays. Quantitative competitive-PCR focuses on this target but design of an internal competitor and validation of amplification efficiencies require a dedicated effort. A recently available approach is based on the 5' nuclease activity of Taq polymerase1 for fragmentation of a dual-labeled fluorogenic hybridization probe2,3. As long as this sequence-specific probe is intact, emission of a reporter dye at its 5' end is quenched by a second fluorescence dye at the 3' end. During extension of PCR, Taq polymerase hydrolyses the probe and releases the reporter dye, the state of which is monitored online by an increase in fluorescence intensity in each tube. As the signal is directly proportional to the amount of amplified copies, valid measurement over a large range of starting quantities is possible and was proven to be a highly reproducible tool with easy handling as compared to conventional PCR cycling4. Absolute quantitation using a standard curve of a target with known quantity is possible, as well as relative quantitation using an appropriate housekeeping gene for internal calibration.

The need for quantitative mRNA measurement is one prerequisite when addressing cellular events in intact tissue; another is the accurate access of cells to be examined. However, commonly applied mRNA extraction procedures are performed with homogenized tissue and reflect an average expression, not allowing analysis of a single cell type. The use of microdissection and micromanipulation techniques to obtain pure cell populations have so far been used only for DNA analysis5,6,7, while mRNA studies required the use of at least small tissue fragments still containing heterogeneous cell types. Our group recently showed that UV-microbeam-assisted cell picking is an appropriate morphology-based technique for contamination-free isolation of cells from complex tissue sections, and mRNA examination of "picked" cardiac neurons was demonstrated on a qualitative basis8. We now describe an approach that allows quantitation of mRNA in a few isotypic cells within intact tissue using a combination of laser-assisted cell picking and real-time PCR.

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Figure 1: Original recording of real-time PCR using mRNA extracted from lavaged alveolar macrophages.
Figure 2: a, Upregulation of TNF-α mRNA in alveolar macrophages exposed to LPS/IFN-γ in vitro.
Figure 3: Laser-assisted cell picking of alveolar macrophages from hematoxylin-stained frozen lung tissue sections.

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Acknowledgements

We thank M.M. Stein and C. Ebner for technical assistance, W. H. Gerlich and R. Snipes for critical reading of the manuscript and R. Oehlmann (PE Applied Biosystems) for support using the ABI Prism 7700 SDS. This study was supported by the DFG (SFB 547 "cardiopulmonary vascular system", project Z1).

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Authors and Affiliations

  1. Department of Pathology, Justus-Liebig-Universität Gieβen, Langhansstr. 10, Gieβen, 35392, Germany

    Ludger Fink, Leander Ermert, Ulrich Stahl & Rainer M. Bohle

  2. Department of Internal Medicine, Justus-Liebig-Universität Gieβen, Germany

    Ludger Fink, Werner Seeger & Friedrich Grimminger

  3. Department of Pediatrics, Justus-Liebig-Universität Gieβen, Germany

    Jörg Hänze

  4. Institute for Anatomy and Cell Biology, Justus-Liebig-Universität Gieβen, Germany

    Wolfgang Kummer

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  1. Ludger Fink
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Correspondence to Ludger Fink.

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Fink, L., Seeger, W., Ermert, L. et al. Real-time quantitative RT–PCR after laser-assisted cell picking . Nat Med 4, 1329–1333 (1998). https://doi.org/10.1038/3327

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