Hybridization enhancement using microfluidic planetary centrifugal mixing.
DNA microarrays produce their greatest sensitivities when hybridized using concentrated samples and effective mixing; however, these goals have proved elusive to combine. If samples are diluted enough to fill larger chambers, then mixing works well using either pumping or gravity with rotation, although sensitivities will suffer. Various techniques for mixing concentrated samples in small thin chambers have been proposed; however, they often leave streaks or scars, and their reusable components require careful cleaning. Here we introduce a versatile new microfluidics platform, a two-axis centrifuge whose fluidic chambers rotate in a planetary relationship to a radial gravitational field. This paradigm readily overcomes surface and viscous forces even in chambers only 50 microm thin. Thin chambers obviate the need for sample dilution and increase sensitivities and dynamic ranges 10-fold. In comparisons against conventional mixing using the same 10 microg of starting total RNA on 22 000-probe arrays, 10 000 more usable signals rose above the noise. In other experiments, planetary mixing was able to produce comparable results while using only one-tenth the starting sample. The benefits of planetary mixing include sample conservation, shorter hybridizations, less reliance on amplification, and the ability to quantify many gene signals otherwise obscured by noise.
Bynum
Magdalena A
MA
Agilent Laboratories, Agilent Technologies, 3500 Deer Creek Road, Palo Alto, California 94304, USA. maggie_bynum@agilent.com
Gordon
Gary B
GB
D000818:Animals; D002498:Centrifugation / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D006321:Heart; D008099:Liver / Q000737:chemistry; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D012313:RNA / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D013499:Surface Properties
D012313:RNA