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

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 ttests), 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 c.a. 1 3 10 4 to 500 3 10 4 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. '

Strand displacement in DNA-based materials systems

“…First, one can oen rely on qualitative monitoring of DNA-mediated colloidal aggregation and dispersion using the naked eye to observe the visible color changes arising from plasmonic effects between neighboring colloidal gold particles. In addition to the ability to qualitatively monitor the behavior of DNA-functionalized particles, quantitative characterization tools ranging from ow cytometry [108][109][110][111][112] to optical trapping 23,113 can be employed to assess hybridization activity on colloidal surfaces. Thus, for DNA-conjugated particle systems, strand displacement has profound implications for dynamic tuning of the magnitude and range of colloidal interactions in order, for example, to reverse aggregation in disordered systems 114,115 or to adjust the lattice spacing in ordered structures.…”

Biosensors for disease biomarker detection

“…Particle-based biosensors are emerging as a feasible alternative to current two-dimensional molecular diagnostics Trau, 2007, 2009;Marcon et al , 2008Marcon et al , , 2009Marcon et al , , 2010Chen et al , 2009;Surawski et al , 2008Surawski et al , , 2009Corrie et al , 2008aCorrie et al , , 2009Johnston et al , 2006Johnston et al , , 2008Vignali, 2000;Bogue, 2005;Kellar and Iannone, 2002;Morgan et al , 2004;Nolan and Mandy, 2006;Nolan and Sklar, 2002). Sample preparation and assay chemistries used by particle-based assays have been adapted from those used for two-dimensional microarray technologies (Nolan and Mandy, 2006;Nolan and Sklar, 2002).…”

Development of a multiplexed bead-based assay for detection of DNA methylation in cancer-related genes

“…File 3 (ESIw) contains an example spreadsheet for an 8-plex assay. For detailed information on the cytometric procedures, refer to Corrie et al 39 T-tests were used to confirm the distinct MF values for both the model system and cell-line DNA experiments. A threshold value of a = 0.01 was used to determine significance, such that if the p-value o a, the null hypothesis was rejected and the combinations of U-and M-probes were considered valid for binary methylation analysis.…”