A simple method for PCR based analyses of immunohistochemically stained, microdissected, formalin fixed, paraffin wax embedded material.
Microdissection was performed on sections cut from formalin fixed, paraffin wax embedded archival material, which had been subjected to conventional immunohistochemistry. Crude DNA extracts, which were obtained from these microdissected samples by a simple microwave step, were then added directly to amplification reactions. Analyses using a range of polymerase chain reaction (PCR) based techniques, including microsatellite repeat polymorphism analysis at the NM23-H1 locus and sequencing of exons 5, 7, and 8 of the p53 gene, were performed successfully. Universal PCR amplification was also carried out on the microdissected material and probes suitable for use in comparative genomic hybridisation (CGH) were obtained in all cases. This technique will enable a range of effective genetic analyses to be carried out on specific subsets of cells that have been characterised previously by immunohistochemistry.
Alcock
H E
HE
Institute for Cancer Studies, Sheffield University Medical School, UK.
Stephenson
T J
TJ
Royds
J A
JA
Hammond
D W
DW
D015179:Colorectal Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D004210:Dissection; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007124:Immunoenzyme Techniques; D019656:Loss of Heterozygosity; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde