2.1. MiRCURY LNA MicroRNA Array
Two versions of miRCURY LNA microRNA arrays are used for miRNA profiling (see the Materials and Methods section for more details). All slides are scanned using an Axon Gene Pix Professional 4200A microarray scanner (Molecular Devices, Sunnyvale, CA, USA), and the images are gridded and analyzed using ImaGene 7.0 software (BioDiscovery Inc., Hawthorne, CA, USA). MiRCURY LNA microRNA Array v7.5.0 (LNAv7 hereafter) is used to profile 359 miRNAs for two HCT-116 cell lines. MiRCURY LNA microRNA Array v9.2 (LNAv9 hereafter) is used to profile 577 miRNAs for 10 osterosarcoma xenograft specimens. On each slide of the LNA arrays, there are four technical replicates for each miRNA. The background signals are estimated by measuring the intensity of the surrounding area (pixels in the local background region) of the corresponding spot masks, and the signal for an miRNA from a specific spot is approximated by the intensity measure from the local signal region. The expression of an miRNA is computed based on the (local) background subtracted signals from the four replicates. All spots are automatically flagged by the image processing software to check the signal quality.
Table 1 shows the five-number summaries of the automatic flags for the spots (probes) for the miRNAs being tested based on thirteen (13) LNAv7 slides and forty-eight (48) LNAv9 slides, respectively. From Table 1, we find the following: Among the 13 LNAv7 arrays, on average, approximately 58% of the probes have reasonably strong signals (not flagged). In the worst case, about 42% of spots are not flagged, and approximately 57% of the spots are low-expressed or missing spots. One slide contains more than 77% of spots with no flags.Among the 48 LNAv9 arrays, on average, less than 20% of the probes have reasonably strong signals, while more than 50% of the probes are empty spots. The best slides have approximately 45% of non-flagged spots, while the non-flagged spots have less than 4% in the worst slide.For both LNAv7 and LNAv9, the proportions of poor spots (background/signal contaminated, high ignored percentage and others) are relatively low.
microarrays-02-00034-t001_Table 1 Table 1  Quality flags with miRCURY LNA arrays (all hsa-miR probes).   In terms of the flagged spots, it looks like LNAv7 arrays have better signal quality than LNAv9 arrays. One potential explanation for the observation is that more weakly or not expressed miRNAs are included in the LNAv9 arrays. To have a closer comparison, we illustrate the summary information for the flags of the 224 miRNAs tested by both LNAv7 and LNAv9 in Table 2. From Table 2, we find that even when comparing the same set of miRNAs among both versions, there was reduced signal in LNAv9 arrays. This difference could also be due to the use of different samples in the experiments: HCT-116 cell lines for LNAv7 and human osterosarcoma xenograft specimens for LNAv9, respectively.
microarrays-02-00034-t002_Table 2 Table 2  Quality flags with miRCURY LNA arrays (overlapped hsa-miR probes).

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