title: Atomic force microscopic detection enabling multiplexed low-cycle-number quantitative polymerase chain reaction for biomarker assays.
abstract: Quantitative polymerase chain reaction is the current "golden standard" for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.
authors.affiliation: Department of Physics, Virginia Commonwealth University , Richmond, Virginia 23284, United States.
authors.forename: Andrey
authors.initials: A
authors.lastname: Mikheikin
authors.forename: Anita
authors.initials: A
authors.lastname: Olsen
authors.forename: Kevin
authors.initials: K
authors.lastname: Leslie
authors.forename: Bud
authors.initials: B
authors.lastname: Mishra
authors.forename: James K
authors.initials: JK
authors.lastname: Gimzewski
authors.forename: Jason
authors.initials: J
authors.lastname: Reed
mesh_terms: D015870:Gene Expression; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics
chemical_list: D012313:RNA