Rolling circle amplification - A new approach to increase sensitivity for immunohistochemistry and flow cytometry

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays . Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this appr oach to routine clinical specimens. Rolling circle amplification (RCA) gene rates a localized signal via an isothermal amplification of an oligonucleot ide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immun ological and molecular probes. RCA-mediated signal amplification was succes sfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracel lular molecules (vimentin and prostate-specific antigen) within a variety o f routinely fixed specimens, as well as samples prepared for flow cytometry . RCA technology, which has an intrinsically wide dynamic range, is a robus t and simple procedure that can provide a universal platform for the locali zation of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emergin g information from genomics and proteomics into cell- and tissue-based anal yses.