NEW mirVana™ miRNA Reference Panel v9.1
A Positive Control Reference Sample for miRNA Analysis

Small RNA discovery and investigation is rapidly expanding. Researchers primarily focusing on the discovery and characterization of new miRNAs have requested a well-characterized positive control reference sample for miRNA analysis. In response to these requests, Applied Biosystems has created a synthetic, equimolar pool of all known miRNAs from the human, mouse, and rat genomes.

A Multifunctional Positive Control for miRNA Analyses
The mirVana™ miRNA Reference Panel v9.1 is a single mixture of RNA oligonucleotides representing an extensive collection of human, mouse and rat miRNAs listed in miRBase Sequence Database Version 9.1 hosted by the Sanger Institute [1]. The panel comprises unmodified, HPLC-purified, single-stranded RNA oligonucleotides. Here we describe the performance characteristics and potential uses of the mirVana miRNA Reference Panel v9.1 for human, mouse, and rat mature miRNA research projects.

The mirVana miRNA Reference Panel provides a linear dose response when assayed by either real-time PCR or microarray analysis. This was demonstrated by titrating known amounts of a subpool of 96 miRNA members of the mirVana miRNA Reference Panel v9.1 and regression analysis by these two methods. See the sidebar, Linearity of Dose Response: A Comparison Using Two Analysis Platforms, for details and data from this experiment.

Applications
The mirVana miRNA Reference Panel v9.1 has several important applications for performing precise analytical measurements and is part of the RNA Controls portfolio that Applied Biosystems offers. Here are some examples:

Sample preparation. Applied Biosystems scientists have demonstrated the usefulness of the mirVana miRNA Reference Panel v9.1 as an in-process control for sample preparation using the flashPAGE™ Fractionator Apparatus and other mirVana sample preparation methods (e.g., mirVana miRNA Isolation Kit; data not shown).

Array hybridization control. The data in the sidebar, Linearity of Dose Response: A Comparison on Two Analysis Platforms, show the use of this reference panel as an array hybridization control for the mirVana miRNA Bioarrays. Likewise, it can be used in conjunction with the mirVana miRNA Probe Set v2 for spotted arrays to control for printing variation. This application will allow for tighter control of array processing and data normalization methods.

Real-time PCR. The mirVana miRNA Reference Panel v9.1 will also be useful as a quantitative standard for real-time PCR based on the careful mixing of equimolar amounts of each oligonucleotide in the panel. This will allow for absolute quantification of molar or mass amounts of miRNAs in an unknown sample relative to a standard curve generated using this product (Figure 1).

Figure 1. Average CT Values for Standard Curve Using mirVana™ miRNA Reference Panel v9.1. Serial, 10-fold dilutions of the mirVana miRNA Reference Panel v9.1 were prepared using 1000, 100, 10, and 1 attomole of each miRNA per RT-PCR reaction. Standard curves were generated using these dilutions of the Reference Panel for a subset of 22 miRNAs. Average CT values for each molar input are shown (±1 standard deviation). Although researchers will not use a mean standard curve for quantification of an unknown sample, this example shows the linearity and dynamic range of the Reference Panel for a small number of TaqMan® MicroRNA Assays.

Small RNA cloning and sequence analysis. This product can also be used as a positive control for small RNA cloning and sequence analysis. Researchers may wish to assay a radiolabeled probe for a target miRNA within miRBase v9.1 against this panel to serve as a positive control in Northern blotting.

More to Come
This is the first commercial release of a reference sample for small RNAs. The mirVana miRNA Reference Panel v9.1 may be used as a positive control for any miRNA research applications focusing on mature human, mouse, or rat miRNAs contained in the v9.1 release of the miRBase collection at the Sanger Institute. Going forward, we plan to add additional annotated sequences and other classes of small RNAs to this portfolio of RNA controls and further define the range of applications of this product.

Reference
1. Access miRBase at: http://microrna.sanger.ac.uk/sequences

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

Figure 2. miRNA Pooling for Latin Square Mixtures. In Latin square 1, 12 miRNAs across a row, each having a common mass input, were pooled. Eight row-pools were then combined, thus mixing eight mass inputs to create each mixture. In Latin square 2, 16 miRNAs of a common mass input from two columns were pooled. Six column-pools were combined, thus mixing six mass inputs to create each mixture. The 20 pg and 200 pg data points were omitted from Latin Square 2.

Figure 3. Linear Response of 96 Synthetic miRNAs in Real-Time PCR (TaqMan® MicroRNA Assays). Mean CT values from triplicate real-time PCRs were plotted, unnormalized, against mass input on a log10 scale. Each data point represents the mean of all 96 miRNAs at the stated mass input. Error bars represent the standard error of the mean (SEM). Data for 0 pg inputs from both studies are omitted from the chart. LS1 = Latin Square 1; LS2 = Latin Square 2.

Figure 4. Concordance of Latin Square Data Between Two Platforms. Unnormalized median array signal intensities or mean CT values were compared using Spotfire® software (Spotfire, Inc.) to determine the concordance of the two platforms as a function of mass input. Each data point is the average of two replicates. Four representative miRNAs are shown. Each data point represents the median array signal and mean CT value for the stated miRNA at a single mass input. The negative slope results from the inverse relationship between CT and copy number in real-time PCR. LS2 = Latin Square 2.