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

Heidi S. Erickson; Paul S. Albert; John W. Gillespie; Jaime Rodriguez-Canales; W. Marston Linehan; Peter A. Pinto; Rodrigo F. Chuaqui; Michael R. Emmert-Buck

doi:10.1038/nprot.2009.61

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

Heidi S. Erickson, Paul S. Albert, John W. Gillespie, Jaime Rodriguez-Canales, W. Marston Linehan, Peter A. Pinto, Rodrigo F. Chuaqui, Michael R. Emmert-Buck

Translational Molecular Pathology

Research output: Contribution to journal › Article › peer-review

80 Scopus citations

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.

Original language	English (US)
Pages (from-to)	902-922
Number of pages	21
Journal	Nature protocols
Volume	4
Issue number	6
DOIs	https://doi.org/10.1038/nprot.2009.61
State	Published - 2009

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1038/nprot.2009.61

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title = "Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples",

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.",

author = "Erickson, {Heidi S.} and Albert, {Paul S.} and Gillespie, {John W.} and Jaime Rodriguez-Canales and {Marston Linehan}, W. and Pinto, {Peter A.} and Chuaqui, {Rodrigo F.} and Emmert-Buck, {Michael R.}",

note = "Funding Information: ACKNOWLEDGMENTS 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.",

year = "2009",

doi = "10.1038/nprot.2009.61",

language = "English (US)",

volume = "4",

pages = "902--922",

journal = "Nature protocols",

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AU - Erickson, Heidi S.

AU - Albert, Paul S.

AU - Gillespie, John W.

AU - Rodriguez-Canales, Jaime

AU - Marston Linehan, W.

AU - Pinto, Peter A.

AU - Chuaqui, Rodrigo F.

AU - Emmert-Buck, Michael R.

N1 - Funding Information: ACKNOWLEDGMENTS 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.

PY - 2009

Y1 - 2009

N2 - 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.

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

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ASJC Scopus subject areas

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