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miRCURY LNA miRNA ISH Optimization Kits (FFPE)

For setup and optimization of miRNA in situ hybridization (ISH) experiments
  • Convenient setup and optimization of miRNA in situ hybridization (ISH) experiments
  • Superior sensitivity and specificity with double DIG-labeled detection probes and optimized reagents
  • Highly robust and supports high-throughput and individual miRNA localization studies
  • Fast and easy one-day protocol
  • High flexibility with no advanced instruments required
  • Validated in a wide range of tissues, including FFPE samples

The miRCURY LNA miRNA ISH Optimization Kits (FFPE) offer all the essential reagents to set up and optimize miRNA in situ hybridization (ISH) experiments in the lab. These kits include double DIG-labeled miRCURY LNA miRNA Detection Probes, ISH buffer and reagents.

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Cat No./ID: 339450
miRCURY LNA miRNA ISH Buffer Set (FFPE)
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2x Formamide-free miRNA ISH buffer, Proteinase K
Cat No./ID: 339451
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 1
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miRCURY LNA miRNA ISH Optimization Kit 1 (miR-1); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-1p
Cat No./ID: 339452
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 2
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miRCURY LNA miRNA ISH Optimization Kit 2 (miR-21); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-21-5p
Cat No./ID: 339453
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 3
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miRCURY LNA miRNA ISH Optimization Kit 3 (miR-122); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-122p-5p
Cat No./ID: 339454
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 4
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miRCURY LNA miRNA ISH Optimization Kit 4 (miR-124); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-124-3p
Cat No./ID: 339455
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 5
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miRCURY LNA miRNA ISH Optimization Kit 5 (miR-126); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-126-3p
Cat No./ID: 339456
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 7
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miRCURY LNA miRNA ISH Optimization Kit 7 (miR-145); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-145-5p
Cat No./ID: 339457
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 8
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miRCURY LNA miRNA ISH Optimization Kit 8 (miR-205); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-205-5p
Cat No./ID: 339458
miRCURY LNA miRNA ISH Optimization Kit (FFPE) 9
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miRCURY LNA miRNA ISH Optimization Kit 9 (miR-223); includes controls, buffer and proteinase K for 400 slides; detects hsa-miR-223-3p
Cat No./ID: 339459
miRCURY LNA miRNA ISH Buffer and Controls
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Scramble miRNA Negative Control Probe, U6 snRNA Positive Control Probe, 2x Formamide-free miRNA ISH buffer, Proteinase K; for 400 slides
miRCURY LNA miRNA ISH Optimization Kits (FFPE) are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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miR-1 detection in adult mouse heart.
The miRCURY LNA miRNA ISH Optimization Kit 1 was used to detect miR-1 in mouse heart FFPE tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-21 detection in colon adenocarcinoma.
The miRCURY LNA miRNA ISH Optimization Kit 2 was used to detect miR-21 in colon adenocarcinoma FFPE tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-122 detection in mouse liver.
The miRCURY LNA miRNA ISH Optimization Kit 3 was used to detect miR-122 in normal murine liver FFPE tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-124 detection in human brain.
The miRCURY LNA miRNA ISH Optimization Kit 4 was used to detect miR-124 in human brain lesion FFPE tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-126 detection in colon wall.
The miRCURY LNA miRNA ISH Optimization Kit 5 was used to detect miR-126 in FFPE samples with inflamed colon tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-145 detection in human colon.
The miRCURY LNA miRNA ISH Optimization Kit 7 was used to detect miR-145 in FFPE tissue of human colon wall with underlying muscle layers. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-205 detection in human breast carcinoma.
The miRCURY LNA miRNA ISH Optimization Kit 8 was used to detect miR-205 in human breast carcinoma FFPE tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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miR-223 detection in esophagus cancer.
The miRCURY LNA miRNA ISH Optimization Kit 9 was used to detect miR-223 in FFPE samples with human esophagus cancer. Staining was performed with NBT-BCIP (blue). Sections were counterstained with Nuclear Red.
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Principle of the miRCURY LNA miRNA ISH Optimization Kit (FFPE).
First, the tissue is "opened" using Protease K. In the hybridization step, the DIG-labeled LNA probe binds specifically to its target miRNA. Alkaline phosphatase (AP)-conjugated anti-DIG antibodies are then added. This step is followed by NBT-BCIP development and optional counterstaining with Nuclear Red.
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miRCURY LNA miRNA ISH Kit chooser.
Choosing the appropriate miRCURY LNA miRNA ISH Optimization Kit. The table indicates the tissue(s) in which each of the kits has been validated.
Performance
Double DIG-labeled probes for superior sensitivity
The miRCURY LNA miRNA ISH Optimization Kits (FFPE) use double DIG-labeled miRCURY LNA miRNA Detection Probes. These probes offer exceptional sensitivity and specificity, so they even can be used to detect low-abundance miRNAs.

All of the kits have been thoroughly validated for use with a wide range of tissues to ensure the highest level of performance (see table below).

Description of the miRCURY LNA miRNA ISH Optimization Kits (FFPE)
Product Unique probe Examples of miR-expressing cells Data figure
Kit 1 miR-1 Skeletal muscle cells, myocytes, myocardiocytes miR-1 detection in adult mouse heart
Kit 2 miR-21 Cancer-associated fibroblasts, some immune cells miR-21 detection in colon adenocarcinoma
Kit 3 miR-122 Hepatocytes miR-122 detection in mouse liver
Kit 4 miR-124 Neurons miR-124 detection in human brain
Kit 5 miR-126 Endothelial cells miR-126 detection in colon wall
Kit 7 miR-145 Smooth muscle, vascular smooth muscle (pericytes), myoepithelial cells miR-145 detection in human colon
Kit 8 miR-205 Basal keratinocytes, myoepithelium, SCC cancer cells miR-205 detection in human breast carcinoma
Kit 9 miR-223 Myeloid, granulocytic, and monocytic compartments of the hematopoietic system miR-223 detection in esophagus cancer


The ideal way to optimize miRNA ISH experiments
All of the miRCURY LNA miRNA ISH Optimization Kits (FFPE) include a double DIG-labeled scrambled probe and a 5’ DIG-labeled U6 control probe. The U6 probe targets U6 snRNA, a small RNA expressed in most tissues, making it an ideal positive control probe in the initial stages of setting up an ISH experiment. The scrambled probe is used as a negative control probe.

In addition, a kit-specific, double DIG-labeled miRCURY LNA miRNA Detection Probe is provided with each kit. These probes can be used as positive control probes in the experiment, once the initial optimization has been performed.

A solution for many problems

The miRCURY LNA miRNA ISH Optimization Kits (FFPE) are very flexible, offering solutions for many different experimental setups without the need for advanced instruments. Some applications include:

  • Study of cellular and sub-cellular miRNA localization
  • Examination of spatial miRNA expression
  • miRNA expression analysis during developmental processes
  • ISH analysis of FFPE samples
  • Biomarker discovery in FFPE samples
  • Potential miRNA ISH in other species where the miRNA target sequence for the kit-specific LNA probe is conserved
  • Evaluation of results from functional studies
Principle
Features
The miRCURY LNA miRNA ISH Optimization Kit (FFPE) is a great tool to help you get started with or to optimize miRNA in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue samples.

Based on the highly successful double (5’ and 3’) DIG-labeled miRCURY LNA miRNA Detection Probes, the kits provide the sensitivity and specificity needed to detect miRNAs (see figure Principle of the miRCURY LNA miRNA ISH Optimization Kit). In addition, the reagents included have been specifically adapted for use with LNA-enhanced probes in FFPE tissue sections.

Coverage
Eight different miRCURY LNA miRNA ISH Optimization Kits are available. Each kit comes with positive and negative control probes, hybridization buffer and Proteinase K. In addition, a unique, tissue-specific miRCURY LNA miRNA Detection Probe is included in each kit.

Each kit includes:
  • Unique miRNA LNA probe (double DIG-labeled, kit specific)
  • Scrambled LNA probe (double DIG-labeled, negative control)
  • U6 LNA probe (5’ DIG-labeled, positive control)
  • Hybridization buffer (2x, formamide-free)
  • Proteinase K
The unique miRNA LNA probes have been validated in a variety of tissues and are therefore ideal for optimizing ISH experimental settings. For recommendations on which kit to choose based on tissue type, refer to the figure miRCURY LNA miRNA ISH Kit chooser.
Procedure
The One-Day miRNA ISH Protocol included in the kit handbook has been thoroughly tested and optimized. Our scientists have eliminated several steps normally associated with ISH, such as pre-hybridization, post-fixation and acetylation, making the protocol very robust, easy to optimize and ideal for high-throughput use. Both the hybridization and washing steps of the One-Day miRNA ISH Protocol are performed using completely formamide-free buffers, which makes working with the kits less cumbersome.
Applications
The miRCURY LNA miRNA ISH Optimization Kits (FFPE) 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 it ideal for use in both high-throughput ISH analysis and localization studies of individual miRNAs.
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