MicroRNA microarray specificity and quantification. (a) Specificity was assayed using a set of 23 microRNA and mismatched probe pairs (two mismatches). Average mean spot intensities from 10 independent hybridizations at 50°C were added to give a total signal for probes corresponding to a given microRNA as well as for probes with two mismatches to the microRNA. Mismatch probe design and sequences are described in Additional data file 6. A specificity index was calculated as 100 × (probe signal - mismatched probe signal)/probe signal. Melting temperatures for the microRNA probes were calculated using the nearest neighbors method . The specificity index is plotted against the calculated melting temperature for each microRNA probe pair. Correlation of melting temperature and specificity index is significant (p = 0.004, Student's t-test). (b) Number of mismatches between microRNAs based on all known mouse microRNAs (the miRNA Registry 3.0 ). Each microRNA was aligned pairwise to every other microRNA and was assigned to the group (number of mismatches) corresponding to the least number of mismatches to another microRNA. (c) Quantification of microarray data using three synthetic RNAs: syn1, syn2 and syn3. Each data point is the average of two independent labelling/hybridization reactions. Probes for the three synthetic RNAs were printed in quintuplicate on the microarray. RNAs were used at 0.025, 0.1, 0.375, 0.75, 2.5, 5 and 10 fmoles. For comparison, the background signal of the array is shown. For more details, see Additional data file 5.