NEW Ambion® mirVana™ miRNA Reference Panel v9.1
A Validated Positive Control and Quantitative Standard for miRNA Studies

The Ambion® mirVana™ miRNA Reference Panel v9.1 was designed as a positive control and quality control sample for any miRNA research application focusing on mature human, mouse, and rat miRNAs. As summarized here, to confirm the functionality of each synthetic oligonucleotide in the Reference Panel, extensive validation experiments using mirVana miRNA Bioarrays and available TaqMan® MicroRNA Assays were conducted. Consistent with Applied Biosystems quality control, three independent manufacturing lots were compared for consistent and reproducible performance.

mirVana miRNA Reference Panel v9.1
The mirVana miRNA Reference Panel is a mixture of over 730 synthetic RNA oligonucleotides present at equimolar concentration and is suitable for use as a positive control and/or quantitative standard in many miRNA applications, including array analysis (e.g., mirVana miRNA Bioarrays), real-time PCR (e.g., TaqMan® MicroRNA Assays), sample preparation (e.g., flashPAGE™ Fractionator and mirVana miRNA Isolation Kit), and small RNA cloning/sequence analysis. One miRNA Reference Panel is sufficient to serve as a positive control for 100 TaqMan Real-Time Assays or 20 mirVana miRNA Bioarrays.

Reference Panel Validation
Three manufacturing lots of mirVana miRNA Reference Panel were validated independently using both Ambion mirVana Bioarrays and TaqMan MicroRNA Assays. There were 619 TaqMan MicroRNA Assays available at the time of the validation study (almost 700 mammalian TaqMan MicroRNA Assays are now available). In each case, duplicate microarrays and TaqMan reactions were run for 6 arrays and 3714 assays, respectively. The data illustrate the high reproducibility of the Reference Panel for both real-time PCR and microarray analysis (Figure 1). Figure 2 shows the distribution of the CT values (mean CT value=13.7) across all 619 assays. The broad distribution of CT values shows a similar trend as the broad slopes of the dose response curves of the probes on the Bioarray.

Figure 1. High Correlation and Reproducibility of Three Independent Lots of the mirVana™ miRNA Reference Panel. (A) Comparison of mean CT values for 617 TaqMan® MicroRNA Assays between two different lots of the mirVana miRNA Reference Panel using 1 fmol per reaction are shown. Two assays were removed from the analysis because their mean CT value was 40. (B) High correlation (R2) and reproducibility (slope) are observed with the Reference Panel in both real-time PCR (TaqMan MicroRNA Assays) and array analysis (mirVana miRNA Bioarray).

Figure 2. Distribution of Raw CT Values for Each Sequence in the mirVana™ miRNA Reference Panel. The distribution of mean CT values for 617 miRNAs in the Reference Panel is shown. Plotted CT values are the average of 6 replicates (3 lots, with 2 replicates per lot).

This extensive validation of the Reference Panel illustrates our commitment to developing and manufacturing the highest quality reference samples. Taken together, this comprehensive data set shows that each miRNA sequence in the mirVana miRNA Reference Panel has been functionally validated either by real-time PCR or microarray analysis.

Additional Data
To see additional data from Ambion mirVana miRNA Bioarray experiments, see article, The Newest Tool for miRNA Expression Profiling in Human, Mouse, and Rat Samples. A list of the assays used, their CT values, and the best-fit line data for the probes on the mirVana Bioarray v9.2 are available online.

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