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Custom microRNA Detection Probes for ISH

Take advantage of Exiqon’s expertise in designing LNA™-enhanced detection probes targeting custom RNA sequences.

  • Unmatched detection of small RNA by in situ hybridization and Northern blotting
  • Superior specificity and sensitivity
  • Designed by our expert team for optimal LNA™ content and positioning
  • Minimal secondary structure and self-annealing
  • Use custom probes as positive and negative controls for microRNA target specificity
  • Use custom probes to detect presence of the in vivo microRNA inhibitor within the target tissue
  • Ideal for standardized protocols in automated High-Throughput ISH
  • Fast and easy workflow using the One-day microRNA ISH protocol and buffer kits

LNA™-enhanced custom probes for optimal detection of any microRNA

Custom miRCURY LNA™ microRNA Detection probes are available for specific detection of novel microRNAs, microRNAs not covered by any pre-designed miRCURY LNA™ microRNA Detection probe, or other short ncRNA sequences. These custom microRNA detection probes are designed by our technical support scientists applying the same design rules used with our pre-designed microRNA detection probes.

The custom probes are designed with optimal LNA™ positioning for achieving high sequence specificity, low secondary structure and minimal self-annealing. An advantageous high melting temperature (Tm) in the range of 82-86°C results in probes with high binding affinity and high signal-to-noise ratios, ideal for implementation of robust protocols, applicable in automated in situ hybridization and automated image analysis.

Content

The custom miRCURY LNA™ microRNA Detection probes are available in two different amounts (1 nmole and 10 nmole), and with a variety of 3’-end, 5’-end, or double (3’ and 5’) labeling options: DIG, biotin, fluorescein (FAM), amino, TYE™ 563 and TYE™ 665. “Ready-to-label” probes designed for custom labeling are also available. For ordering, price information and requests for alternative labels, please contact us.

Higher sensitivity with double-labeled probes

Double (3’ and 5’) DIG- or double- fluorescein (FAM)-labeled probes offer substantially higher sensitivity than single labeled probes and are the most sensitive detection probes available. A cooperative effect of the two labels results in greatly increased signal-to-noise ratio (up to 10-fold higher) which means that even low abundance microRNAs can be reliably detected.

We recommend double DIG or double fluorescein labeling for optimal results. miRCURY LNA™ microRNA Detection probes are available with the following double labels: double-DIG, double-fluorescein (FAM), double-TYE™ 563, double-TYE™ 665, and double-biotin.

Applicable in IHC and pathology labs

The miRCURY LNA™ microRNA Detection Probes are used with great success in a variety of samples. These include whole mounts, single cells and sections from fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues, e.g. biobank archived material. As such the probes enable in situ hybridization in routine clinical FFPE samples.

The miRCURY LNA™ microRNA Detection Probes are compatible with a large range of in situ hybridization protocols, whether for chromogenic or fluorescent detection. Furthermore, our ISH protocols can be used together with IHC to perform double stains. The combination of LNA™ for increased affinity and Tm-normalization means that all probes perform optimally under the same conditions. This facilitates very robust standardized protocols, which makes the miRCURY LNA™ microRNA Detection Probes ideal for ISH automation.

Recommended protocol and buffers for FFPE samples

For microRNA in situ hybridization experiments in FFPE tissue sections Exiqon recommend using the One-day microRNA ISH protocol, see the ISH manual for double-labeled probes. The protocol is optimized for the miRCURY LNA™ microRNA Detection Probes and contains fewer steps, resulting in a very fast procedure comparable in time to IHC staining protocols. The miRCURY LNA™ microRNA ISH buffer set (FFPE) is developed specifically for use with the miRCURY LNA™ microRNA Detection Probes.

Note

Before submitting your order, please check if a pre-designed miRCURY LNA™ microRNA Detection probe targeting your sequence is available. Pre-designed miRCURY LNA™ microRNA Detection Probes are available for most microRNA sequences annotated in miRBase.

Unmatched specificity and sensitivity

The high affinity and discrimination of the miRCURY LNA™ microRNA Detection Probes result in sensitive detection of microRNAs. Specific in situ detection of microRNA is possible in whole mounts, single cells, frozen samples and in formalin-fixed, paraffin-embedded tissue sections, including archived biobank samples. The optimized positioning of LNATM within the detection probes substantially reduces background staining.

For maximum sensitivity and detection of even lowly expressed targets, we recommend double labeled miRCURY LNA™ microRNA Detection Probes. To enable multiplexing, a range of haptens are available, to visualize the target microRNA with different combinations of FISH and/or chromogenic ISH.

Get optimal customized ISH Control probes

Custom miRCURY LNA™ microRNA Detection Probes are ideal for the detection of any small RNA sequences, but they also serve as valuable controls in ISH experiments. Our LNA™ experts can design custom probes in a number of different ways in order to confirm signal specificity of the in situ hybridization reaction. As with any experiment, interpretation of the results is only as good as the controls. For in situ hybridization there is no single best control, so it is preferable to have as many controls as possible, thus building confidence in the expression pattern.

Below please find examples of Custom miRCURY LNA™ microRNA Detection Probes, designed specifically for use as positive and negative control probes.

Ideal as Mismatch Negative Control probes

Mismatch probes are custom designed specifically to be used alongside the target probe. A mismatch probe differs by 1-3 nucleotides towards the target. Take full advantage of the LNA™ technology. Our LNA™ design experts will identify the optimal number of mismatches and the LNA™ positions, to reach a Tm value close to that of the target probe.

Using the same in situ protocol on serial sections, one should obtain little or no stain with the mismatch negative control probe. An example of a custom mismatch negative control probe can be seen in Figure 1f from the publication Nielsen et al., 2011. High levels of microRNA-21 in the stroma of colorectal cancers predict short disease-free survival in stage II colon cancer patients. Clin Exp Metastasis. 28(1):27-38. PMID: 21069438.

Target-specific positive Control probes

The short length of LNA™ probes enables alternative probes to be designed that target slightly different regions, shifted by a few nucleotides – even within the short sequence of a mature microRNA (Figure 2). Obtaining the same hybridization pattern with different probes targeting the same microRNA is a valuable positive control in ISH experiments. Our LNA™ experts custom design alternative probes with LNA™ positions and Tm value similar to that of the target probe.

Pre-microRNA detection probes

Another valuable positive control probe to rule out non-specific cross hybridization is a loop-directed probe, designed by our LNA™ experts to bind to the loop sequence of the precursor microRNA (pre-microRNA) (Figure 2). This probe should confirm the same hybridization pattern as the target probe that binds to the mature microRNA sequence. However, the expression level of the pre-microRNA is likely to be lower than that of the mature microRNA.

An example of target-specific positive control probes, including a loop-directed probe, can be seen in Figure 1 from the publication Rask et al., 2011. High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS. 119(10):663-73. PMID: 21917003.

Further information

View experimental data on the miRCURY LNA™ microRNA Detection Probes for

Figure 1 Binding sites of four different LNA™ probes targeting the mature and precursor microRNA-21
Binding sites of four different LNA™ probes targeting the mature and precursor microRNA-21. (Click to learn more)
Liming Luan

MicroRNAs in Skin


"Exiqon is one of the leading suppliers for miRNA-related products."

Dr. Liming Luan at Vanderbilt University Medical Center in Nashville studies the role of microRNAs in normal and pathological conditions of the skin.

Read full story...

Mircea Ivan

MicroRNAs in Hypoxia


"I was intrigued by this interesting technology, which is very different from other available technologies."

Dr. Mircea Ivan at Indiana University School of Medicine studies the role of microRNAs in hypoxia. Here he describes why his lab chose Exiqon's detection probes.

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Ronald H. A. Plasterk

MicroRNA Involvment in Embryo Development


"We got some beautiful data and spectacular images. We generated a complete catalogue of images showing the temporal and spatial expression patterns of 115 conserved microRNAs in zebrafish embryos."

Prof Ronald H. A. Plasterk, Hubrecht Laboratory, The Netherlands.

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Dylan Sweetman

MicroRNAs in Muscle Cell Differentiation


"Following the success of LNA™ probes as demonstrated in Plasterk's lab, we decided to take the same approach."

Dr. Dylan Sweetman is working in Dr. Andrea Münsterberg’s group at the University of East Anglia, School of Biological Sciences. The group investigates cellular and molecular mechanisms that underlie embryonic development.

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Parker Antin

MicroRNAs and Early Embryonic Development


"LNA™ technology is superior to any other for the detection of small RNA species."

Dr. Parker Antin's lab at the University of Arizona runs a large scale in situ hybridization database project to determine the expression patterns for all differentially expressed genes in the chicken embryo. Find out what the challenges were with this huge project and how they were overcome.

Read full story...

This is a custom LNA™ product. Please contact us for further information and ordering.
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