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ViewRNA miRNA ISH Cell Assay- How It Works

mRNA and miRNA in situ hybridization assay workflow

The ViewRNA miRNA ISH Cell Assay is based on patent-pending probe design and bDNA signal amplification technology.

  • For the mRNA in situ hybridization, a target specific probe set, containing 40 oligo probes, hybridizes to the target mRNA as 20 oligo pairs. For clarity, only two oligo pairs are shown per target mRNA (Figure 1). Each oligo pair forms a required platform for assembly of the signal amplification structure (tree) through a series of sequential hybridization steps. Each fully assembled structure, covers a space of 40-50 nt of your target mRNA, and has the capacity for 400-fold signal amplification. Therefore, a typical mRNA probe set (containing 20 oligo pairs) has the capacity to generate 8,000-fold signal amplification. Independent but compatible signal amplification systems enable simultaneous detection of up to 2 target mRNAs.
  • For the miRNA in situ hybridization, a target specific probe set contains one oligonucleotide pair that covers the entire mature miRNA sequence (20-23 nt). A fully assembled structure has the capacity for 400-fold signal amplification. Fast Red Substrate is used for sensitive visualization of the miRNA target.
  • The ViewRNA assay can be performed on cover slips processed in a 24-well plate or in a 96-well plate. The assay has four main steps: sample preparation, target hybridization with probes, signal amplification using bDNA technology, and detection. At the end of the assay the processed cover slips are mounted on standard microscope slides for imaging or if the 96-well plate was used, no further processing for imaging is required.

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    Specificity of the miRNA probe in the miRNA in situ hybridization assay

    miR-548h & miR-548i in situ hybridization probes

    The ability of the ViewRNA miRNA ISH Cell Assay to distinguish highly homologous targets with a 3-base difference was evaluated in situ with synthetic miRNAs transfected in HeLa cells. Very low levels of non-specific signal were observed for many miRNAs differing by three bases (Figure 1, Table I). Read More …

    Specificity of the mRNA probe in the mRNA in situ hybridization assay

    target specific probe sets

    A highly specific and sensitive signal can be achieved by using target-specific probe sets comprised of 20 oligonucleotide pairs. Read More ...