Calibration of a custom designed microarray. Cottus

We designed a custom expression 8x15 k microarray for Cottus fishes based on transcriptome sequencing. It is a known fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2011."Transcriptome changes after genome wide admixture in invasive sculpins" Molecular Ecology; no doi yet) for more information. Overall design: Labeled cRNA was prepared from 3 Cottus fish. These 3 fish were representative of species (C.rhenanus, C.perifretum, invasive hybrid species) used in another experiment. An calibration pool was prepared from equimolar amounts of this cRNA. Increasing amounts of labeled cRNA (75 ng, 150 ng, 300 ng, 600 ng, 1200 ng, 2400 ng, 4800 ng), corresponding to (1/8, 1/4, 1/2, 1, 2, 4 and 8 times the recommended amount of 600 ng) were hybridized to 7 microarrays (one microarray per dilution-step). The change in observed signal intensity in relation to the change in amount of labeled cRNA was used to infer the target-binding behavior of the individual probes. This information was extracted, to be used for a normalization procedure in another experiment with the same microarray (see Czypionka et al. 2011."Transcriptome changes after genome wide admixture in invasive sculpins" Molecular Ecology; no doi yet)