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

  1. Author:
    Erickson, H. S.
    Albert, P. S.
    Gillespie, J. W.
    Rodriguez-Canales, J.
    Linehan, W. M.
    Pinto, P. A.
    Chuaqui, R. F.
    Emmert-Buck, M. R.

  2. Author Address

    Erickson, Heidi S.; Rodriguez-Canales, Jaime, Chuaqui, Rodrigo F.; Emmert-Buck, Michael R.] NCI, Pathogenet Unit, Pathol Lab, NIH, Bethesda, MD 20892 USA. [Erickson, Heidi S.; Rodriguez-Canales, Jaime, Linehan, W. Marston, Pinto, Peter A.; Chuaqui, Rodrigo F.; Emmert-Buck, Michael R.] NCI, Urol Oncol Branch, NIH, Bethesda, MD 20892 USA. [Albert, Paul S.] NCI, Biometr Res Branch, Div Canc Treatment & Diag, NIH, Bethesda, MD 20892 USA. [Gillespie, John W.] NCI, SAIC Frederick Inc, Frederick, MD 21701 USA.
    1. Year: 2009
    2. Epub Date: 5/21/2009
  2. Journal: Nature Protocols
    1. 4
    2. 6
    3. Pages: 902-922
  4. Type of Article: Article
  5. ISSN: 1754-2189
  1. 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 similar to 16 h.

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  2. DOI: 10.1038/nprot.2009.61
  3. PMID: 19478806

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