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miRCURY LNA miRNA Detection Probes

For ultra-sensitive and specific miRNA detection by in situ hybridization (ISH) or Northern blotting
  • Superior sensitivity and specificity for detecting low-abundance miRNAs
  • Predesigned probes available for all miRNA species
  • A wide selection of available labels enables multiplexing and co-localization
  • Great for standardized protocols for automated, high-throughput ISH
  • Fast and easy workflow using the one-day miRNA ISH protocol
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.

Please contact us for further product information, or technical questions.
Search in GeneGlobe
You can search for the following terms:
  • Entrez Gene IDs (e.g., 835)
  • RefSeq IDs (e.g., NM_032983, NP_116765)
  • Gene symbols (e.g., CASP2)
  • Cat. no. (e.g., SI00299551, QT01342509)
  • Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)
  • CpG loci identification numbers (CG#) (e.g., CG17753661)

Do not enter species information in the Search box. Use the drop-down list to select your species of interest.
When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

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产品 Product no. 货号 目录价:
miRCURY LNA miRNA Detection Probe (1 nmol)
1 nmol miRCURY LNA miRNA Detection Probe with option of different labels, provided in single-tube format
339111 Varies Enter your targets in search box above.
miRCURY LNA miRNA Detection Probe (10 nmol)
10 nmol miRCURY LNA miRNA Detection Probe with option of different labels, provided in single-tube format
339112 Varies Enter your targets in search box above.
miRCURY LNA miRNA ISH Buffer Set (FFPE)
2x Formamide-free miRNA ISH buffer, Proteinase K
339450 339450 Enter your targets in search box above.
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产品 Product no. 货号 目录价:
 
 
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miRCURY LNA miRNA Detection Probes适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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Detection of hsa-miR-127 and hsa-miR-154 by fluorescent in situ hybridization using miRCURY LNA detection probes in cryopreserved bone marrow cells from an acute myeloid leukemia patient.
Panels A and B show the cytoplasmic detection of miR-127 and miR-154, respectively. Panel C shows the nuclear expression of the positive control U6 small RNA as visualized using the miRCURY LNA U6 control probe. No signal was detected when cells were hybridized with the miRCURY LNA scramble probe as a negative control, as seen in panel D. Data used with kind permission from Dr. Silvana Debernardi, Institute of Cancer, London, UK. Original figure appears in Dixon-McIver A, East P, Mein CA, Cazier J-B, Molloy G, et al. (2008) Distinctive Patterns of miRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia. PLoS ONE 3(5): e2141. doi:10.1371/journal.pone.0002141.
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miRNA detection in zebrafish.
Specific detection of miR-122a (top), miR-206 (middle) and miR-124a (bottom) using miRCURY LNA miRNA Detection Probes for in situ hybridization of whole-mount zebrafish embryos. This image is a part of a complete catalog of images showing the temporal and spatial expression patterns of 115 conserved miRNAs in zebrafish embryos. The image was kindly provided by Dr. Ronald Plasterk, Hubrecht Laboratory, The Netherlands.
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miRNA detection in chick.
Specific detection of mir-206 in Gallus gallus embryo using a miRCURY LNA miRNA Detection Probe. mir-206 is expressed in all skeletal muscle cells, appearing at the onset of myogenic cell differentiation. At the pictured stage in embryonic development, mir-206 is detected in the myotomal muscle cells (Ason et al. 2006).
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Comparison of double and single DIG labeling.
hsa-miR-21 was detected in tissue sections using a miRCURY LNA miRNA Detection Probe with a double DIG (5’ and 3’) label at 40 nM (A) or a single 3’ DIG label at 80 nM (B). The double DIG probe gives a more intense signal with lower background, even at a lower probe concentration.
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In situ hybridization using double DIG- and double fluorescein (FAM)-labeled LNA probes in FFPE samples.
The examples show miRCURY LNA miRNA Detection Probes with double DIG label and double FAM label for miR-124 in human normal brain samples, and miR-126 and scramble negative control in human colon cancer samples. The miR-126 and scramble probes show the same area on serial sections. Probes were incubated at 30 nM probe concentration and hybridized at 57ºC. The double FAM-labeled LNA probes show the same signal-to-noise as the double DIG-labeled LNA probes, and can be used independently or in combination to co-localize miRNAs using different hapten-labeled probes or antibodies, in the case of protein co-detection by immunohistochemistry. Images kindly provided by Boye Schnack Nielsen, Manager, Molecular Histology at Bioneer A/S, Hørsholm, Denmark.

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Dual stain in normal mammary gland by ISH and immunohistochemistry (IHC) in a double-fluorescence assay.
Co-localization of miR-205 (pink) and cytokeratin (green) in the normal mammary gland. miR-205 was detected using a double fluorescein (FAM)-labeled miRCURY LNA miRNA Detection Probe, and cytokeratin was detected using a chromogenic secondary antibody. Counterstain: DAPI (blue). Images kindly provided by Boye Schnack Nielsen, Manager, Molecular Histology at Bioneer A/S, Hørsholm, Denmark.
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Dual stain in ductal carcinoma in situ (DCIS) by ISH and immunohistochemistry (IHC) in a double-fluorescence assay.
Co-localization of miR-205 (pink) and cytokeratin (green) in ductal carcinoma in situ (DCIS). miR-205 was detected using a double fluorescein (FAM)-labeled miRCURY LNA miRNA Detection Probe, and cytokeratin was detected using a chromogenic secondary antibody. Counterstain: DAPI (blue). Images kindly provided by Boye Schnack Nielsen, Manager, Molecular Histology at Bioneer A/S, Hørsholm, Denmark.
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LNA probes are superior to DNA probes when it comes to detecting miRNAs.
Two duplicate dilution series of A. thaliana total RNA were hybridized with 32P-labeled DNA and miRCURY LNA miRNA Detection Probes, respectively, for A. thaliana miR-171 and miR-319. From Válóczi et al. 2004, Nucleic Acids Res. e175; reprinted with permission from Oxford University Press.
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LNA probes readily discriminate between single nucleotide differences.
The specificity of the probe was assessed using 32P-labeled perfect match, double mismatch and single mismatch miRCURY LNA miRNA Detection Probes for the detection of miR-171 in A. thaliana flowers (1) and leaves (2). The filters were washed at either low stringency or high stringency. From Válóczi et al. 2004, Nucleic Acids Res. e175; reprinted with permission from Oxford University Press.
Performance
Superior sensitivity and specificity for ISH and Northern blotting
miRCURY 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 signals
Dual-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 staining
A 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 results
The 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).
Principle
Coverage
Predesigned 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 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 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. 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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