Quantitative microarray spot profile optimization: A systematic evaluation of buffer/slide combinations

The non-trivial choice of a good combination of microarray spotting buffer and slide chemistry is critical for the reliability of microarray hybridization experiments. This choice determines the morphology of spots and, moreover, the variance of that morphology. Both strongly affect how well the extracted intensity signals correspond to sample RNA abundances. Comparisons of different buffer/slide combinations, however, have tended to be subjective, and are not suitable for a systematic approach. Spot morphology and its variance were assessed objectively by measuring the average and variance of radial spot pixel intensity profiles. The variances assessed comprised deviations from radial symmetry and spot-to-spot reproducibility, as well as general pin-to-pin differences and slide effects. A quantitative and systematic evaluation of over 24×6 buffer / slide combinations yielded a new protocol giving superior spot profiles with minimal variance whilst also scoring excellently on other technical requirements. The process introduced here can generally be applied to evaluate new materials and technologies as they emerge. Software is available on request from the authors. For an online supplement, please see http://www.inference.phy.cam.ac.uk/dpk20/spotprofiles/. Correspondence: [email protected]