Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms

S. R. Corrie, G. A. Lawrie, B. J. Battersby, K. Ford, A. Rühmann, K. Koehler, D. E. Sabath, M. Trau

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

Abstract

Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of ca. 1 × 104 to 500 × 104 molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories.

Original languageEnglish
Pages (from-to)467-476
Number of pages10
JournalCytometry Part A
Volume73
Issue number5
DOIs
Publication statusPublished - 1 May 2008
Externally publishedYes

Keywords

  • Absolute target quantitation
  • Bead assay
  • Beads
  • Equilibrium constant
  • Flow cytometry
  • Fold-ratio
  • Microspheres
  • Molecular diagnostic
  • Quantitation
  • T-test

Cite this