Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.
Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.
Heinmöller
Ernst
E
Department of Molecular Genetics, City of Hope National Medical Center/Beckman Research Center, Duarte, California 91010-0269, USA.
Liu
Qiang
Q
Sun
Yuan
Y
Schlake
Gudrun
G
Hill
Kathleen A
KA
Weiss
Larry M
LM
Sommer
Steve S
SS
D001940:Breast / Q000166:cytology* / Q000378:metabolism; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D004210:Dissection; D000431:Ethanol; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008846:Micromanipulation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000235:genetics*; D017422:Sequence Analysis, DNA; D016707:Tissue Fixation; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics*
D017931:DNA Primers; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53; D000431:Ethanol; D004247:DNA