2.3. Experimental Validation
The mathematical model was validated using the real-time hybridization experiments performed on Xeotron hybridization station (Xeotron Corp., Houston, TX, USA—now part of Invitrogen). Eighteen targets were designed to complement the 18 probes for the hybridization experiments (Table 1). However, this is not a large enough sample size to make predictions about the effect of individual melting temperatures on hybridization kinetics due to other confounding factors such as mismatches [27]. The targets (45–52 mers) were synthesized and amine modified on the 3′ end by Operon Biotechnologies Inc. (Huntsville, AL, USA - part of MWG-Biotech AG, Ebersberg, Germany). The targets were coupled with Cy3 (Amersham Biosciences, Little Chalfont, United Kingdom, Cat. No. PA23001) and then dissolved in dimethyl sulphoxide (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. D2438 in 75 mM sodium carbonate buffer at pH 9.0). A hybridization buffer containing 35% deionized formamide (Thermo-Fisher Scientific, Waltham, MA, USA), 6x SSPE (pH 6.6; Invitrogen; 1x SSPE contains 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA pH 7.7), and 0.4% Triton X-100 (Sigma-Aldrich) [22]. Several hybridization experiments were performed to assess the effects of temperature, concentration and flow rate. The real-time data images of nucleic acid hybridization were obtained using OSA Reader v1.91 software (IMSTAR, Paris, France) in conjunction with a CCD camera (resolution 10 μm per pixel) attached to a Nikon Eclipse 50i fluorescent microscope (100× magnification; Nikon Instruments, Melville, NY, USA).