For ultra-sensitive and specific detection of novel miRNA sequences by in situ hybridization (ISH) or Northern blotting

Features

Superior sensitivity and specificity for detecting low-abundance miRNAs

Custom-designed probes for targeting novel miRNA sequences

A wide selection of available labels enables multiplexing and co-localization

Ideal for standardized protocols for automated, high-throughput ISH

Fast and easy workflow using the one-day miRNA ISH protocol

Product Details

Custom miRCURY LNA miRNA Detection Probes enable specific detection of novel miRNAs, miRNAs not covered by the predesigned miRCURY LNA miRNA Detection Probes or other short, ncRNA sequences. These custom miRNA detection probes are LNA enhanced and are designed by our technical support scientists, according to the same design rules used for our predesigned miRNA detection probes.

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Performance

Get optimal, customized ISH control probes

Custom miRCURY LNA miRNA Detection Probes are great for detecting any small RNA sequences, and they also serve as valuable controls in ISH experiments. Our LNA experts can design custom probes in a number of different ways so you can confirm signal specificity of your ISH experiment. As with any experiment, interpretation of the results is only as good as the controls. For ISH, there is no single best control, so it is preferable to have as many controls as possible to provide confidence in the observed expression pattern.

Great for use as mismatch negative control probes

Mismatch probes are custom designed specifically for use alongside the target probe. A mismatch probe differs by 1–3 nucleotides towards the target. Our LNA design experts will identify the optimal number of mismatches and LNA positions, to reach a Tm value close to that of the target probe.

Using the same ISH 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 following 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 the design of alternative probes that target slightly different regions, shifted by a few nucleotides, even within the short sequence of a mature miRNA (see figure Binding sites of four different LNA probes targeting the mature and precursor miRNA-21). Obtaining the same hybridization pattern with different probes targeting the same miRNA is a valuable positive control in ISH experiments. Our LNA experts can custom design alternative probes with LNA positions and a Tm value similar to that of the target probe.

Pre-miRNA detection probes

Another valuable positive control probe to rule out non-specific cross hybridization is a loop-directed probe, designed to bind to the loop sequence of the precursor miRNA. This probe should confirm the same hybridization pattern as the target probe that binds to the mature miRNA sequence. However, the expression level of the pre-miRNA is likely to be lower than that of the mature miRNA.

An example of target-specific positive control probes, including a loop-directed probe, can be seen in Figure 1 from the following 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.

Principle

Coverage

Custom miRCURY LNA miRNA Detection Probes are a great choice if a predesigned probe for your miRNA target is not available. The probes are highly suited for use in both in situ hybridization (ISH) and Northern blotting experiments. The probes are available with a selection of 3', 5' or dual (3' and 5') labels: DIG, biotin and fluorescein (FAM). Ready-to-label probes for custom labeling are also available.

One-day miRNA ISH protocol

The one-day miRNA ISH protocol is designed for detection of miRNA in FFPE tissue sections. The protocol uses the non-mammalian hapten digoxigenin (DIG) or fluorescein and has been optimized for a one-day experimental setup. First, miRNAs are demasked using Proteinase K, allowing double-DIG or double-fluorescein labeled LNA probes to hybridize to the miRNA sequence. The DIG or fluorescein haptens are recognized by a specific anti-DIG or anti-fluorescein antibody, respectively, that is directly conjugated to alkaline phosphatase (AP). AP converts the soluble substrates 4-nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3’-indolylphosphate (BCIP) into a dark blue precipitate. A nuclear counterstain is applied to the sections to allow better histological resolution.

Application in IHC and pathology labs and more

miRCURY LNA miRNA Detection Probes are useful in a variety of tissue and cell preparations, including whole mounts, single cells and sections from fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues, such as biobank-archived material. Thus, these probes can be used for routine ISH analysis of FFPE samples. The probes are compatible with a variety of ISH protocols using both chromogenic or fluorescent detection. Furthermore, our ISH protocols can be used together with IHC to perform double stains.

LNA enhancement in the miRCURY LNA miRNA Detection Probes increases probe affinity and provides Tm-normalization, ensuring that all probes perform optimally under the same conditions. This facilitates very robust standardized protocols, making the probes a great tool for use in automated ISH protocols. Plus, the high signal-to-noise ratio facilitates automated image analysis.

Recommendations for FFPE ISH

For miRNA ISH experiments in FFPE tissue sections, we recommend using the one-day miRNA ISH protocol. Refer to the kit handbook if you are using dual-labeled probes. The protocol is optimized for use with the miRCURY LNA miRNA Detection Probes and contains fewer steps, providing a very fast procedure compared with IHC staining protocols. The miRCURY LNA miRNA ISH Buffer Set (FFPE) is developed specifically for use with the miRCURY LNA miRNA Detection Probes.

Procedure

The one-day miRNA ISH protocol minimizes time-consuming optimization steps, enabling fast and optimal miRNA ISH analysis using a colorimetric antibody-based development system for DIG-labeled probes. Each step of the procedure is detailed, including tissue sectioning, recommended selection of miRNA-specific positive and negative control probes, incubation times and temperatures, miRCURY LNA miRNA Detection Probe concentrations and substrate incubations. Refer to the kit handbook for details.

Applications

The miRCURY LNA miRNA Detection Probes can be used for a large number of applications, including cellular and sub-cellular miRNA localization studies and determination of spatial miRNA expression, as well as Northern blotting experiments. The robustness of the ISH procedure makes them a great tool for use in both high-throughput ISH analysis and localization studies of individual miRNAs.