Exiqon - LNA for microRNA research

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Figure 1

Tm-normalized LNA™ capture probes.

Figure 7

Easy assessment of data quality

Figure 8

Excellent spot morphology

Figure 9

High reproducibility

Figure 10

Excellent cross-platform correlation

miRCURY LNA™ microRNA Arrays for expression profiling

At a glance

Sensitivity - microRNA profiling possible from 30 ng total RNA
Specificity - efficient discrimination between closely related microRNA family members
Reproducibility - high reproducibility with 99% correlation between arrays
Dynamic range - greater than 4 orders of magnitude
Diversity - most comprehensive probe set available
Open platform - protocols available for Tecan and MAUI hybridization stations, and for manual hybridization

Product coverage
The miRCURY LNA™ microRNA Array portfolio now consists of two arrays:

The most recent version of the array (v. 10.0 - hsa, mmu & rno array) contains more than 1200 capture probes which cover all human, mouse and rat microRNA sequences annotated in miRBase 10.0. The array also interrogates all microRNAs of the viruses related to these three species.
The 9.2 all species array contains more than 2000 capture probes, making it possible to profile miRBase 9.2 microRNAs from any organism – vertebrates, invertebrates, plants and viruses – and to cross profile between different species.

Both arrays contain approximately 50 miRPlus™ capture probes for detection of microRNAs not included in miRBase. These probes give researches unique access to information about microRNAs otherwise not available.

Table 1. Coverage of miRBAse 10.1

Tm-normalized LNA™ capture probes
Detection of short target sequences, such as microRNAs, is inherently difficult and is complicated by the large variation in base composition of the microRNAs, which range in GC content from 25 to 90% (for human microRNAs). In order to be comparable, highly discriminative, and sensitive, microarray capture probes should optimally have similar melting temperatures (i.e. be Tm-normalized).

However, using pure DNA probes, Tm -normalization compromises probe design because the maximum hybridization temperature will be dictated by the lowest full-length microRNA capture probe duplex Tm on the array, i.e. the Tm of the most AT-rich duplex. In order to adjust the Tm of capture probes targeting the more GC-rich microRNAs (high Tm), significant truncations generating capture probes as short as 8-9 nucleotides, are required.

Such short capture probes present a significant problem because of their poor specificity due to the high frequency of 8-9-meric sequences in the transcriptome. Furthermore, probe sensitivity is reduced due to the short probe length. This means that Tm –normalization of pure DNA capture probes is not possible without compromising specificity and sensitivity of some of the probes.

This paradox is solved by incorporation of LNA™ in the capture probes of the miRCURY LNA™ microRNA Arrays. By adjusting the LNA™ nucleoside content as well as the length of the probes, the capture probes have been Tm -normalized to ensure that all microRNA targets hybridize to the array with equal affinity under high-stringency hybridization conditions. LNA™ capture probes have been designed according to empirically derived algorithms to maximize their affinity and specificity for their microRNA target. Figure 1 illustrates how the Tm of LNA™ capture probes is increased significantly and the Tm range is narrowed significantly compared to DNA probes. This makes miRCURY LNA™ microRNA Arrays superior to DNA-based arrays, especially when it comes to the detection of AT-rich microRNAs (Figure 2).

Sensitivity
The sensitivity of miRCURY LNA™ microRNA Arrays have been assessed by empirically testing 705 human microRNA capture probes using synthetic microRNAs. More than 90% of the LNA™ capture probes on the array have a detection limit of ≤10 amol, enabling microRNA profiling even with very small amounts of total RNA (Figure 3).

Sample input
Without prior knowledge of the microRNA content in the sample, it is recommended to use 250 to 1000 ng total RNA to facilitate the most robust expression profiles. However, the high sensitivity of miRCURY LNA™ microRNA Arrays enables reliable microRNA expression profiles from only 30 ng of total RNA (Figure 4).

Specificity
miRCURY LNA™ microRNA Arrays are highly specific for their microRNA targets. The combination of Tm -normalized LNA™ capture probes and hybridization conditions optimized for high stringency binding allows for accurate detection of microRNA expression and increases the specificity of the capture probes. The optimized LNA™ capture probe design provides superior distinction between closely related microRNAs and will, in most cases, be able to specifically distinguish between microRNAs that differ by only one nucleotide (Figure 5).

Dynamic range
miRCURY LNA™ microRNA Arrays offer superior dynamic range over more than 4 orders of magnitude, ensuring that microRNAs with high and low expression levels will be detected well within the linear detection range (Figure 6).

Improve data quality with spike-ins
All miRCURY LNA™ microRNA Array products contain 10 synthetic spike-in microRNAs that can be detected on the arrays by specifically designed capture probes. When the spike-in microRNAs are added to the labeling reactions before a dual-color array hybridization, the signals from the spike-in capture probes can be used:

as a control for the labeling reaction and hybridization
to calibrate/adjust scanner settings between channels
as a control for the data normalization procedure
to estimate the variance of replicated measurements within arrays
to assess technical variability between different parts of the array

Figure 7 illustrates the position of the 10 spike-in microRNAs in 1 ug total RNA.

Reproducibility
The miRCURY LNA™ Array features very high reproducibility through an optimized manufacturing processes that ensures high quality uniform spots (Figure 8). This results in very low CV values of the four replicate spots as well as excellent inter-slide correlation (Figure 9).

High cross-platform correlation
Data obtained with the miRCURY LNA™ microRNA Array demonstrate excellent cross-platform correlation with data obtained using other profiling technologies. An example of such correlation is demonstrated in Figure 10. A comparison was made of miRCURY LNA™ microRNA Array data and published profiling data of placenta, ovary and liver tissue using small RNA cloning (Landegraf et al., 2007) or real-time PCR (Liang et al., 2007). The RNA used for these studies were from different sources and were, therefore, subjected to significant differences with respect to variations between donors, tissue fractions and sample processing procedures. Moreover, the cloning data from some of the less abundant microRNAs constituted only 15 counts in total from the three different tissues, resulting in some “stochastic noise”. Nevertheless, the hierarchical cluster analysis showed excellent correlation between the three different techniques, resulting in tight tissue clustering independent of platform.