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Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples

Nature Protocols volume 4, pages 902–922 (2009)Cite this article

Abstract

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen-related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires ∼16 h.

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Figure 1: Tissue sectioning and staining flowchart.
Figure 2: Frozen prostate epithelium laser capture microdissection (LCM) procurement sequence.
Figure 3: qRT-PCR from microdissected tissue process flowchart.
Figure 4: Dedicated RT-PCR hood setup.
Figure 5: Example qPCR 96-well setup.
Figure 6: Typical TaqMan qPCR amplification plots for 96-well setup.

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Acknowledgements

The authors appreciatively thank S. Gonzalez and A. Velasco (The Catholic University, Santiago, Chile) for their collaboration in providing the frozen prostate whole-mount tissue blocks used to assess normalization strategies. R.F.C. and M.R.E.-B. are Federal employee inventors on NIH patents covering LCM and xMD technologies and are entitled to receive royalty payments through the NIH Technology Transfer program. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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Authors and Affiliations

  1. Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Heidi S Erickson, Jaime Rodriguez-Canales, Rodrigo F Chuaqui & Michael R Emmert-Buck

  2. Division of Cancer Treatment and Diagnosis, Biometric Research Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Paul S Albert

  3. SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, Maryland, USA ,

    John W Gillespie

  4. Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Maryland, USA

    W Marston Linehan & Peter A Pinto

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  1. Heidi S Erickson
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Correspondence to Michael R Emmert-Buck.

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Erickson, H., Albert, P., Gillespie, J. et al. Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples. Nat Protoc 4, 902–922 (2009). https://doi.org/10.1038/nprot.2009.61

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