miRCURY LNA miRNA Detection Probes
For ultra-sensitive and specific miRNA detection by in situ hybridization (ISH) or Northern blotting
miRCURY LNA miRNA Detection Probes are designed with optimal LNA positioning to have high sequence specificity, low secondary structure and minimal self-annealing. The resulting superior binding affinity lets you specifically and sensitively detect miRNA with an exceptionally high signal-to-noise ratio.
Superior sensitivity and specificity for ISH and Northern blottingmiRCURY LNA miRNA Detection Probes are designed with optimal LNA positioning to achieve high sequence specificity, low secondary structure and minimal self-annealing. This results in superior binding affinity as well as very specific and sensitive detection of miRNA with an exceptionally. The melting temperatures (Tm) of the probe:target duplexes are normalized around 84°C and are typically in the range of 82–86°C, enabling robust protocols that support automated analyses.
A number of peer-reviewed publications have demonstrated the excellent performance of these probes in ISH experiments by showing ultra-specific miRNA expression pattern in zebrafish and chicken embryos (see figures miRNA detection in zebrafish and miRNA detection in chick).
For researchers who wish to detect miRNAs on Northern blots by non-radioactive methods, we recommend the use of double (3' and 5') DIG-labeled or fluorescein probes. miRCURY LNA miRNA Detection Probes offer very high binding affinity, single-nucleotide discrimination and remarkable signal-to-noise ratios, resulting in highly specific and sensitive miRNA detection via Northern blotting. High specificity and sensitivity of the probes makes it possible to reduce Northern blot sample sizes to 1/10 of that required for traditional probes. Moreover, it allows you to reduce exposure times to just a few hours (see figure LNA probes are superior to DNA probes when it comes to detecting miRNAs). miRCURY LNA miRNA Detection Probes enable discrimination of double or even single mismatches (see figure LNA probes readily discriminate between single nucleotide differences).
Dual-labeled probes for even higher signalsDual-labeled probes with DIG or fluorescein offer substantially higher sensitivity than single-labeled probes and are the most sensitive detection probes available. The two labels produce a cooperative effect, greatly increasing the signal-to-noise ratio by up to 10 fold, so even low-abundance miRNAs can be reliably detected (see figure Comparison of double and single DIG labeling). We recommend double DIG or double fluorescein labeling for optimal results. The sensitivity of double-FAM matches that of double-DIG in most cases (see figure In situ hybridization using double DIG- and double fluorescein (FAM)-labeled LNA probes in FFPE samples).
Dual stainingA range of haptens is available for multiplexing applications, enabling visualization of the target miRNA with different combinations of FISH and/or chromogenic ISH. Dual-staining combining miRNA ISH with protein immunohistochemistry (IHC) is highly applicable for combinations of double-FAM probes and chromogenic secondary antibodies (see figures Dual stain in normal mammary gland and Dual stain in ductal carcinoma in situ (DCIS)).
Control probes for validation of resultsThe miRCURY LNA miRNA Detection Control Probes are designed for use with our miRCURY LNA Detection Probes for both ISH and Northern blotting experiments. The control probes are similar in length and LNA design to the detection probes and therefore fall within the same Tm range. The U6 snRNA positive control and the scramble negative control can be used during optimization and troubleshooting of ISH experiments. In addition, these controls can be used to validate experimental results (see figure Detection of hsa-miR-127 and hsa-miR-154).
CoveragePredesigned miRCURY LNA miRNA Detection Probes are available for most known invertebrate, vertebrate and plant miRNAs that are annotated in miRBase and 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. If a predesigned probe for your miRNA target is not available, or if you require a different label, we recommend the Custom miRCURY LNA miRNA Detection Probes.
One-day miRNA ISH protocolThe 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 moremiRCURY 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 ISHFor 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.
The 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.
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. The robustness of the procedure makes them a great tool for use in both high-throughput ISH analysis and localization studies of individual miRNAs.
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