Quantitative microarray spot profile optimization: A systematic evaluation of buffer/slide combinationsD P Kreil1, R P Auburn, L A Meadows, S Russell, G Micklem
1ISMB03@Kreil.Org, University of Cambridge
The non-trivial choice of a good combination of microarray spotting
buffer and substrate 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
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 24x6 buffer/slide
combinations yielded a new protocol giving superior spot
profiles with minimal variance whilst also scoring excellently on
other technical considerations, like strong signal intensity, low
carry-over, low evaporation rate,and lack of rush.
Clearly, this is an optimization problem in a very high dimensional
space, and results strongly depend on many other parameters, like
probe concentration or probe length distribution.
The process introduced here, however, can generally be applied to
explore this space and evaluate new materials and technologies as
The present study is proof of concept work. Specific results for
80ng/Ál cDNA, and for oligo probes will be published elsewhere
(submitted; please request a preprint).