Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens.
We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.
Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
Chikako
C
Uneyama
Makoto
M
Shibutani
Naoya
N
Masutomi
Hironori
H
Takagi
Masao
M
Hirose
D019342:Acetic Acid; D000818:Animals; D002452:Cell Count; D002531:Cerebellum / Q000737:chemistry; D002540:Cerebral Cortex / Q000737:chemistry; D002725:Chloroform; D004247:DNA / Q000032:analysis*; D005404:Fixatives; D008099:Liver / Q000737:chemistry; D008297:Male; D000432:Methanol; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D016610:Tissue Embedding
D005404:Fixatives; C046544:methacarn; D002725:Chloroform; D004247:DNA; D019342:Acetic Acid; D000432:Methanol