Microarray results are susceptible to variation introduced from multiple sources during the course of assay methodologies employed with this technology. A commonly overlooked parameter which may significantly impact results is the choice of chamber/vessel for performing microarray incubations and washes. Chambers which allow for active mixing can significantly improve microarray data. Grace Bio-Labs offers ProPlate® incubation chambers which allow for continuous mixing during microarray assays and hybridizations – significantly decreasing variability and increasing sensitivity while minimizing reagent requirements. The use of these chambers significantly reduces microarray variability (CVs ~ 10%) compared to conventional methods (CVs ~ 35%) and allows researchers to interpret their data with the utmost confidence.
Figure 1. 3-D representations and typical uniformities from microarray spots obtained after (A) incubations performed in a ProPlate® Chamber allowing for active mixing versus (B) incubations performed under a glass coverslip providing no mixing. Incubations were performed for 1 hour with anti-IgG-TRITC and -Cy5 at room temperature.
Figure 2. Data show the (i) Intra-Spot variation (pixel-to-pixel), (ii) Intra-Slide variation (spot-to-spot, 10 mm between spot groups), and (iii) Inter-Slide variation (between spots from 4 arrays). All data were obtained from spots deposited on ONCYTE SuperNOVA porous nitrocellulose slides at protein concentration of 1 mg/ml (within the linear dynamic range of binding and detection). Fluorescence data are normalized and background-subtracted, collected at 532 nm from spotted goat IgG assayed with rabbit anti-goat IgG-TRITC. Assay volume under the cover slip was 50 µl (1:25,000 Dilution) and in the ProPlate was 300 µl (1:150,000 Dilution). Intra-Spot CV data are pixel variation per spot, N = 64 spot replicates (over 4 microarrays). Intra-Slide CV data are spot variation per array, N = 4 microarrays (mean of N = 16 spot replicates/array). Inter-slide data were obtained from 4 separate microarray slides.
