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.