Methacarn fixation for genomic DNA analysis in microdissected cells.
We have found methacarn, a non-crosslinking protein-precipitating fixative, to be useful for the analysis of DNA from microdissected specimens of wax-embedded tissue. In this chapter, we present the procedure regarding genomic DNA analysis in methacarn-fixed wax-embedded microdissected rat tissue. Using nested polymerase chain reaction (PCR), and a rapid extraction procedure, fragments of DNA up to 2.8 kb in size can be amplified from a 1 x 1 mm area of a 10-microm-thick tissue section. Target fragments of about 500 bp can be amplified from a single cell, but 10-20 cells are necessary for practical detection by nested PCR. Although tissue staining with hematoxylin and eosin inhibits the PCR, amplification of about 500-bp fragments is successful with 150-270 cells by single-step PCR. Immunostaining results in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, fragments of about 180 bp can be amplified with 150-270 cells by single-step PCR. These features demonstrate the suitability of methacarn-fixed wax-embedded tissue for practical genomic DNA analysis in terms of tissue handling, extraction efficiency, and satisfactory PCR results.
Shibutani
Makoto
M
Division of Pathology, National Institute of Health Sciences, Tokyo, Japan.
Uneyama
Chikako
C
D019342:Acetic Acid; D000818:Animals; D002725:Chloroform; D004273:DNA, Neoplasm / Q000032:analysis*; D005404:Fixatives; D016678:Genome; D007834:Lasers; D000432:Methanol; D042282:Microdissection / Q000379:methods*; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D016707:Tissue Fixation / Q000379:methods*
D004273:DNA, Neoplasm; D005404:Fixatives; C046544:methacarn; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol