Exiqon - LNA for microRNA research

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Figure 1

Get access to the unique miRPlus™ microRNAs

Table 1

Coverage of miRBase 12.0

Figure 2

Figure 1. Sensitivity of less than 1 amol & Data from 30 ng sample

Tm-normalized LNA™ capture probes.

Figure 8

Easy assessment of data quality

Figure 9

Excellent spot morphology

Figure 10

High reproducibility

Figure 11

Excellent cross-platform correlation

miRCURY LNA™ microRNA Arrays for expression profiling

At a glance

435 miRPlus™ microRNAs - access to unique human microRNAs
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
We currently offer two miRCURY LNA™ microRNA Arrays:

V. 11.0 - hsa, mmu & rno array: An array specific for human, mouse and rat microRNAs that contains more than 1700 capture probes, covering all microRNAs annotated in miRBase 11.0, as well as all viral microRNAs, related to these species. The coverage to the latest miRBase v.12.0 is 98%, 97% and 100% for human, mouse and rat, respectively. (See Table 1)
In addition, this array contains capture probes for 435 new miRPlus™ human microRNAs. They are proprietary microRNAs not included in miRBase. The significance of these sequences is underscored by the fact that 30 % of the currently annotated human microRNAs, were available in our miRPlus™ range of products prior to publication in miRBase.
The 9.2 all species array contains more than 2000 capture probes, making it possible to profile miRBase 9.2 microRNAs from any organism – vertebrate, invertebrate, plant and virus – and to cross profile between species.

miRCURY LNA™ microRNA Array - access to unique human microRNAs
Human miRPlus™ microRNAs are proprietary human microRNAs that Exiqon have identified by cloning and sequencing. miRPlus™ microRNAs can be detected using the miRCURY LNA™ microRNA arrays (figure 1).
The first miRPlus™ capture probes were included on the miRCURY LNA™ microRNA array in May 2006 and until now we have had 274 miRPlus™ capture probes on various versions of the miRCURY LNA™ microRNA array. The relevance of the miRPlus™ microRNAs is given by the fact that 236 of 274 miRPLus™ microRNAs now are annotated in miRBase v.12.0.

In other words, almost 30% of the annotated human microRNAs have been on the miRCURY LNA™ Arrays before they were in miRBase.

This clearly shows the high quality of our miRPlus™ microRNAs and that it is yet another benefit for researchers when using our miRCURY LNA™ microRNA arrays.

435 new human miRPlus™ capture probes on current miRCURY LNA™microRNA Array
The capture probes for 435 miRPlus™ microRNAs are included on the miRCURY LNA™ microRNA Array, v11.0 – hsa, mmu & rno. Thereby, the miRCURY LNA™ microRNA Array can identify almost 1300 human microRNAs and 80 human virus microRNAs.
This is by far the highest number of human microRNAs any microarray supplier can detect.

847 Human mature microRNAs
80 Human virus mature microRNAs
435 Human miRPlus™ mature microRNAs

The miRPlus™ sequences are proprietary microRNA sequences that have been identified by cloning and sequencing in human disease or normal tissues. To be include on the miRCURY LNA™ microRNA arrays, they are carefully selected by use of specific criteria such as that the sequences must be found in a number of clones and they must be significant different to miRBase annotated sequences. Thus, the miRPlus sequences are truly different to annotated microRNA sequences and will give researches unique access to information about microRNAs otherwise not available.

The miRPlus sequences are normally submitted to miRBase within 6-12 months, but researches can get access to the sequences by signing a non-disclosure agreement.

Figure 1 illustrates that the new 435 miRPLus™ microRNAs are differential expressed in 6 different human tissues.

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 2 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 3).

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 4).

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 5).

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 6).

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 7).

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 8 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 9). This results in very low CV values of the four replicate spots as well as excellent inter-slide correlation (Figure 10).

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 11. 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.