What is RNAscope®?

RNAscope® is a novel multiplex nucleic acid in situ hybridization technology, based on ACD’s unique, patent-pending probe design and signal amplification methodology. It is the only clinically effective in situ RNA detection assay available today.

 

RNAscope® Features

 

• Fast assay development – Three week turnaround for any new target.
• Ultimate sensitivity – Single molecule detection in situ.
• Exquisite specificity – Can distinguish highly homologous genes.
• Multiplexing capability – Simultaneous detection of multiple targets.
• Quantitative – Each detected target can be quantified on per cell basis.
• Colormetric or fluorescent readout – Under bright field or fluorescent microscope.
• Applicable to all sample types – Exfoliated cells, blood and FFPE specimen.
• Speed and throughput – Fully automated, walk away assay.

 

How it works (click diagram to enlarge)

 

 

The assay consists of target probes and a signal amplification system composed of preamplifiers (PreAMP), amplifiers (AMP), and label probes.

 

Step 1:	Cells in clinical specimen are fixed and permeablized using protease to allow for probe access.
Step 2:	A set of target probes is hybridized to the target RNA of interest. The target probes are standard oligonucleotides that are designed to hybridize as pairs with each pair creating a binding site for a preamplifier.
Step 3:	The preamplifier is hybridized to the target probes at a temperature that favors hybridization to target probe pairs, but not individual target probes. This ensures that if unpaired target probes hybridize non-specifically to a non-specific RNA, no signal amplification will occur. Then, the amplifier is hybridized to the preamplifier. Finally, the label probe, which is conjugated to either a chromogenic or fluorescent molecule, is hybridized to the amplifier.
Step 4:	The stained samples can then be visualized under a standard brightfield or fluorescent microscope.

 

On average, each set of target probes spans an approximately 1kb region of the target RNA and hybridizes to 20 preamplifiers. Each preamplifier can hybridize to 20 amplifiers and each amplifier can hybridize to 20 label probes. For a fluorescent conjugation, this results in over 8,000 fluorescent molecules spanning just 1kb of RNA, which is readily visible using a standard fluorescent microscope.

 

It is possible to increase the signal intensity further still by substituting an AP or a HRP moiety for the fluorescent molecule on the label probe. This allows for a colorimetric reaction that leaves visible colored dots at the enzymatic reaction site. Furthermore, multiple distinct amplification systems have been built that do not cross-react with each other and recognize unique sequences on different sets of target probes allowing for the simultaneous detection of multiple RNA targets.