Search:        >> Advanced Search
Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takagi, H. Articles by Hirose, M. Search for Related Content PubMed PubMed Citation Articles by Takagi, H. Articles by Hirose, M. Social Bookmarking                      What's this?

Microdissected Region-specific Gene Expression Analysis with Methacarn-fixed, Paraffin-embedded Tissues by Real-time RT-PCR

Hironori Takagi, Makoto Shibutani, Natsumi Kato, Haruka Fujita, Kyoung-Youl Lee, Shu Takigami, Kunitoshi Mitsumori and Masao Hirose

Division of Pathology (HT,MS,NK,HF,K-YL,ST,MH), National Institute of Health Sciences, Tokyo; United Graduate School of Veterinary Sciences (HT,MS,KM), Gifu University, Gifu; and Laboratory of Veterinary Pathology (KM), Tokyo University of Agriculture and Technology, Tokyo, Japan

Correspondence to: Dr. M. Shibutani, Div. of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: shibutan{at}nihs.go.jp

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1–1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, 200 cells were necessary for accurate measurement in 10-µm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens. (J Histochem Cytochem 52:903–913, 2004)

Key Words: methacarn • paraffin-embedded tissue • mRNA expression • real-time RT-PCR • microdissection • hematoxylin staining

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. Delfour, P. Roger, C. Bret, M.-L. Berthe, P. Rochaix, N. Kalfa, P. Raynaud, F. Bibeau, T. Maudelonde, and N. Boulle RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells J. Mol. Diagn., May 1, 2006; 8(2): 157 - 169. [Abstract] [Full Text] [PDF]

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2004