BaseScope Assay: Detect RNA exon junctions and short target sequences like never before
Friday, Dec 16, 2016

BaseScope Assay:

Detect RNA exon junctions and short target sequences like never before

Advanced Cell Diagnostics

Based on the established RNAscope® technology (Branched DNA signal amplification technology), ACD has developed the new BaseScope Assay to provide new data dimensions and unique insight into biological mechanisms. The BaseScope™ Assay enables applications such as the detection of exon junctions in FFPE tissue with morphological context and the ability to visualize gene expression for short RNA target sequences (as short as 50bp).

Working principle of Branched DNA signal amplification technology




click to enlarge


The Branched DNA detection system makes use of double Z probe to enhance both sensitivity and specificity.  Each Z target probe contains three elements: The lower region is complementary to the target RNA and is selected for target specific hybridization and uniform hybridization properties. A spacer sequence links the lower region to an upper region.
The two adjacent upper regions from a double Z target probe forms a 28 base binding site for the pre-amplifier. Two independent Z probes, designed as probe pairs, need to hybridize to the target sequence in tandem in order to enable binding of the pre-amplifier. A single Z probe hybridization onto a non-specific RNA target can happen, but the resulting hybridization of the pre-amplifier onto the upper region of a single Z will be unstable and therefore will be removed during the wash steps. This design ensures a low background noise level.


BaseScope Assay vs RNAscope Assay



Application of BaseScope Assay:

1. Exon Junction Detection



Detection of exon junctions in single cells within the tissue environment is possible by using only one ZZ probe uniquely designed on the specific exon junction of interest. For an example of the detection of a sequence with specific exon skipping, METΔ14 (Frampton GM et al, Cancer Discovery, 2015; Awad MM et al, J Clin Oncol, 2016) where 3 probes are designed (image 1): one control probe designed for an exon junction present in all MET transcripts (exon junction 12/13), one probe designed for the exon junction with exon 14 (exon junction 14/15), and one probe designed for the exon junction with exon 14 skipping (exon junction 13/15). Figure 2 shows an example of the BaseScope™ assay used for the detection of METΔ14 in cell lines.

2. Short Target Sequences

The BaseScope™ assay can be used for the detection of short target sequences (figure 1) with morphological context, such as the CDR sequence for T cell receptor (TCR) clones (figure 2), snoRNA, pre-miRNA, or gene therapy. It can also be used to analyze highly homologous gene sequences, partially degraded RNAs (i.e., RNA extracted after decalcification from bone samples), or transiently expressed RNAs.

Learn more about the fundamental RNAscope Technology, click here

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