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Methacarn Fixation for Genomic DNA Analysis in Microdissected Cells
- Source :
- Laser Capture Microdissection ISBN: 9781588292605
- Publication Year :
- 2005
- Publisher :
- Humana Press, 2005.
-
Abstract
- 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.
Details
- ISBN :
- 978-1-58829-260-5
- ISBNs :
- 9781588292605
- Database :
- OpenAIRE
- Journal :
- Laser Capture Microdissection ISBN: 9781588292605
- Accession number :
- edsair.doi...........91dfb69d562a3a81f38c5b6a381c5b18