Single cell PCR from archival stained bone marrow slides: a method for molecular diagnosis and characterization.
Molecular analysis of isolated single cells is a powerful tool for clarifying issues of cell origin and clonality. Previous reports have described PCR amplifications from total DNA and RNA extracted from archival bone marrow and peripheral blood smears and have also shown the feasibility of amplifications from single cells, microdissected from stained histological sections. In this study, a method is described for performing PCR from morphologically defined single cells isolated from archival May-Gruenwald-Giemsa-stained bone-marrow and blood smears. Using three DNA extraction procedures, the organic lysis showed reproducible high efficiencies of amplifications. With this method, we were able to amplify long range amplicons up to 14.5 kb from mitochondrial DNA as well as PCR products of conventional length. The usability of such products for molecular diagnosis is demonstrated by restriction fragment length polymorphism (RFLP)characterization of a mitochondrial disorder. In conclusion, this method has the power to perform molecular diagnosis and characterization of diseases on the single cell level, and should provide valuable information to aid disease treatment and prognosis of hematological disorders.
Zanssen
Stefanie
S
Department of Biochemistry, Medical Faculty, RWTH Aachen, Aachen, Germany. sz2106@columbia.edu
D000328:Adult; D000368:Aged; D000740:Anemia / Q000175:diagnosis* / Q000235:genetics; D001109:Archives; D001854:Bone Marrow Cells / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004272:DNA, Mitochondrial / Q000032:analysis*; D004210:Dissection / Q000379:methods; D005260:Female; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000175:diagnosis* / Q000235:genetics; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D004272:DNA, Mitochondrial; D004247:DNA