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MicroRNA expression profiling of human breast cancer identifies new markers of tumour subtype

Experimental design Bead-based flow-cytometric miRNA profiling is relatively novel, with only few publications utilizing the platform to date [56, 97]. We therefore designed the experiment in a manner that allowed us to investigate technical effects and assess the reproducibility of measurements for a given probe or target sample. As described in [56] probes were coupled to colour-coded polystyrene beads allowing the simultaneous detection of about 90 different target oligonucleotides. To obtain expression profiles for 309 miRNAs we created four distinct sets of bead-coupled miRNA probes. To assess the reproducibility of measurements from distinct bead sets, twelve spare colour-coded beads were selected to carry the same probe in each bead set. These probes consisted of six replicated miRNA probes (let-7a, let-7b, miR-24, miR-26a, miR-181a, miR-224) and six control probes for synthetic oligonucleotides. To assess the reproducibility of sample profiles, 16 out of the 137 samples were selected for replication. 15 samples were hybridized in triplicate and one sample in duplicate, resulting in a total of 168 samples. The 168 x 4 hybridizations were carried out on eight 96-well microtiter plates, using two plates for each bead set (Additional data file 2; panel A). Samples were separated into two groups and, for a given bead set, hybridizations were allocated between plates accordingly. Each of the two groups included at least one replication for each of the 16 replicated samples. Low-level analysis Median fluorescence intensity (MFI) values smaller than a threshold of 1 were set equal to 1. The data were then transformed by taking logs (base 2) to achieve approximate independence of signal intensity and variance. As described previously, we observed some signal for probes in blank control wells (median log 2 MFI 3.9) (Additional data file 3; panel A). To reduce the amount of noise due to probes for miRNAs which were absent in our samples, we performed filtering by requiring each probe to exceed a log 2 MFI of 6 in at least one sample. This value was based on the distribution of signal levels detected in the blank control wells and by considering the number of detected probes for a range of cutoff values (Additional data file 3; panel B). Samples with low mean intensity were more strongly affected by noise. This was confirmed by considering pairwise correlations of the technical replicate samples over a range of intensities (Additional data file 3; panels C, D). Reduced reproducibility was observed …