NEW Ambion® mirVana™ miRNA Bioarray v9.2
The Newest Tool for miRNA Expression Profiling in Human, Mouse, and Rat Samples

• Preprinted arrays for miRNA expression profiling
• Reliable CodeLink™ technology (now supplied by Applied Microarrays) delivers superior linearity, high sensitivity, and low background
• Probes target a comprehensive set of human, mouse, and rat mature miRNAs from miRBase Sequence Database version 9.2, Ambi-miR™ Molecules, and controls

Comprehensive Content
The new Ambion® mirVana™ miRNA Bioarray v9.2 is the latest version of a high quality microarray composed of probes targeting a comprehensive set of human, mouse, and rat miRNAs from miRBase Sequence Database version 9.2 (Figure 1). The Bioarray v9.2 has the same footprint as its predecessor (mirVana miRNA Bioarray V2), with content in the same location in both versions. Bioarray v9.2 contains the same exclusive set of Ambi-miR™ Molecules and control probes present on Bioarray V2:

• The 153 Ambi-miR Molecules are novel miRNAs identified through bioinformatic prediction, cloning, and detection in human total RNA samples.

• One positive control provided in the mirVana miRNA Labeling Kit is used to assess intra-array variability and enable alignment during array scanning. Ten probes are positive controls for use with the mirVana miRNA Bioarray Spike-In Controls to monitor the workflow. (Spike-In Controls consist of synthetic RNA oligonucleotides premixed at concentrations spanning the signal dynamic range of the Bioarrays.)

• 11 negative control sequences have been bioinformatically designed to avoid hybridization to known human, mouse, or rat targets.

Figure 1. Summary of mirVana™ miRNA Bioarray v9.2 Content. Bioarray content includes probes for an extensive collection of human, mouse, and rat miRNAs in miRBase Version v9.2, as well as probes for 153 novel human miRNAs known as Ambi-miR™ Molecules.

Comparison of Bioarray v9.2 With Bioarray V2
As shown here (Figures 2–5), Bioarray v9.2 maintains the same high performance and data quality as the previous version, so that the results from both Bioarrays are compatible.

Control Sample. Bioarray performance was assessed using the mirVana miRNA Reference Panel, a synthetic, equimolar pool of known mature miRNAs from human, mouse, and rat genomes (see article, A Validated Positive Control and Quantitative Standard for miRNA Studies). Figure 2 shows the correlation of the two Bioarray designs using the Reference Panel at 2 concentrations. The data illustrate the high reproducibility of the system.

Figure 2. Correlation of mirVana™ miRNA Bioarray V2 and v9.2 using the mirVana™ miRNA Reference Panel. The mirVana miRNA Reference Panel (0.5 or 5 fmol) was labeled and hybridized to three Bioarray V2s and six Bioarray v9.2s from three lots. Median signal intensity was calculated and log2 transformed. No normalization of signal intensity was performed. Signal intensities below 50 were removed from the analysis. The correlation was performed using 424 miRNAs.

Mixed Tissue Sample. Figure 3 summarizes the results when a mixed tissue sample (human bladder, human lung, and mouse embryo total RNA) is hybridized to Bioarray v9.2 and Bioarray V2. The correlation, using only miRNAs common to both versions, was 0.998 (slope=0.988). In addition, 87 of 251 new human and mouse miRNAs on Bioarray v9.2 were present in this sample. An miRNA is considered present if (Median Signal Intensity–Average Negative Controls)/Standard Deviation of the Negative Controls >3.

Figure 3. High Correlation and Reproducibility of mirVana™ miRNA Bioarrays (v9.2 vs. V2). Comparison of signal intensities from independent arrays demonstrates high array-to-array and lot-to-lot reproducibility. The same labeled sample [10 µg of human bladder, human lung, and mouse embryo RNA (1:1:1 ratio)] was hybridized to three Bioarray V2s and six Bioarray v9.2s from three lots. The median signal intensities of the replicates were averaged and log2 transformed. The signal intensities were not normalized. The data shown represents correlation of sequences present in both versions and has a minimum signal intensity of 50 (n=310). The correlations among the lots were all 0.99, with slopes ranging from 0.96 to 1.02.

Sensitivity of Bioarray v9.2
Titration experiments using the Reference Panel and replicate arrays measured the dose response curve (0.1–2 fmol) for each probe on the Bioarray. A best fit correlation was performed between median signal intensity and molar input of target. Figure 4 shows representative dose response curves for three miRNAs, in which the signal dose response correlation is >99%. While the three miRNA targets demonstrate a strong linear relationship between signal and molar input, the magnitude of response (i.e., slope) differed greatly.

Figure 4. Dose Response Curves for Three Representative miRNAs. Data representing median signal intensity (unnormalized—background subtracted signal only) for each miRNA is plotted as a function of molar concentration. Blue=mmu-miR-383, green=hsa-miR-325, orange=hsa-miR-132. The corresponding slopes are 20.9, 12.2, and 5.7, respectively.

Figure 5A shows the distribution of linear correlation values of dose response curves as a function of slope for all Bioarray probes. The median R² was 0.991, with >90% probes having an R² >0.971. Figure 5B shows the boxplot distribution of R2 values for dose response curves of 731 probes. Two probes gave no measurable signal, even at the highest molar input, and were not included in the analysis.

Figure 5. Distribution of Slopes from Dose Response Curves for Each miRNA in the mirVana™ miRNA Reference Panel Plotted as a Function of R2 for Linear Fit of the Median Signal Intensity and as a Function of Relative Molar Input. (A) The distributions of slopes and the corresponding R2 values for 731 miRNAs are shown. Plotted values were determined from two replicate arrays for each molar input. The boxplot shown on the Y axis (B: blow up of boxplot.) indicates the median median R2 of the data, along with the distribution of the few outlier probes that showed poor performance. The median R2 value is 0.991. The inter-quartile range (sides of the box) and upper 10% and lower 10% quantile are shown. Two miRNAs, hsa-miR-135b and hsa-miR-544, gave no detectable signal on the array and were removed from the analysis.

The Ambion mirVana miRNA Array System
mirVana miRNA Bioarrays do not require processing before hybridization. They are designed for use with control, hybridization, and wash components in the mirVana miRNA Bioarray Essentials Kit and with samples prepared using the mirVana miRNA Labeling Kit. Together these products comprise the mirVana Array System, a platform for miRNA expression profiling that produces sensitive, accurate, and highly reproducible data.

Scientific Contributors
Diane Ilsley, Luming Qu, Andrew Lemire, Penn Whitley, and Robert Setterquist • Applied Biosystems, Austin, TX