Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.
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-microm-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.
Takagi
Hironori
H
Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Shibutani
Makoto
M
Kato
Natsumi
N
Fujita
Haruka
H
Lee
Kyoung-Youl
KY
Takigami
Shu
S
Mitsumori
Kunitoshi
K
Hirose
Masao
M
D019342:Acetic Acid; D000818:Animals; D000831:Animals, Newborn; D002452:Cell Count; D002725:Chloroform; D004396:Coloring Agents; D005404:Fixatives; D005629:Frozen Sections; D020869:Gene Expression Profiling / Q000379:methods*; D006416:Hematoxylin; D000432:Methanol; D042282:Microdissection; D010232:Paraffin; D012313:RNA / Q000032:analysis* / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016610:Tissue Embedding
D004396:Coloring Agents; D005404:Fixatives; D012333:RNA, Messenger; C046544:methacarn; D012313:RNA; D002725:Chloroform; D010232:Paraffin; D019342:Acetic Acid; D000432:Methanol; D006416:Hematoxylin