Description
How RNA FISH Assays works using branched DNA (bDNA) technology
Target hybridization
Traditional FISH techniques use large oligonucleotide sequences labeled with one to five fluorophores and are limited by high background and low sensitivity due to non-specific binding and insufficient signal amplification. With bDNA technology, a target specific probe set of approximately 20 short oligonucleotide pairs hybridize to the specific regions across the target RNA sequence of interest. Subsequent signal amplification steps require that each oligonucleotide pair bind to the target RNA in adjacent positions, providing specificity to the target, as well as an available docking site for other oligonucleotides to hybridize. Additional hybridization steps eventually result in a fluorescently labeled ‘hybridization tree’ that provides single RNA molecule detection by imaging or flow cytometry. Four types of Fluorescent Probe Sets are currently available for ViewRNA Cell or PrimeFlow RNA detection, using various Alexa Fluor dyes with distinct emission spectras. When multiplexing more than one RNA target in a single sample, each Probe Set must be a unique type to differentiate its signal from the others. For ViewRNA ISH Tissue Assays there are only two types of probe sets available that are detectable by fluorescence or chemiluminescence using Fast Red or Fast Blue substrates.
Signal amplification
Signal amplification using bDNA technology is achieved through a series of sequential hybridization steps, which result in the formation of a tree-like structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes to form the “trunk” of the tree. Multiple Amplifier molecules hybridize to their respective Pre-amplifier to create the “branches”. Finally, multiple Label Probes hybridize to the Amplifiers to form the “leaves” of the tree. A fully assembled signal amplification tree contains 400 Label Probe binding sites. If all target-specific oligonucleotides in a 20-oligonucleotide-pair Probe Set bind to the target RNA transcript, an 8,000-fold amplification can be achieved.
Select the appropriate assay for your study
| ViewRNA ISH Cell Assays | ViewRNA Cell Plus Assay | ViewRNA Tissue Assay | ViewRNA Tissue Fluorescence Assay | PrimeFlow RNA Assay | ||
|---|---|---|---|---|---|---|
| mRNA ISH | High-content ISH | |||||
| Cell samples | Cultured cells (adherent or suspension) | Cultured cells (adherent or suspension) | Cultured cells (adherent or suspension) | FFPE tissue sections | FFPE tissue sections
Cryopreserved tissue sections Cell culture cells (adherent) |
Single cell suspensions (primary or cultured) |
| Multiplexing | Up to four RNA targets | Up to four RNA targets | Up to four RNA targets (including miRNA) and one or more protein targets, or any combination of the above | Up to two RNA targets (including miRNA) | Up to four RNA targets including microRNA | Up to four RNA targets (including miRNA) |
| Antibody compatibility | No | No | Yes | No | No | Yes |
| Detection signal | Fluorescence
|
Fluorescence
|
Fluorescence
|
Fluorescence or colorimetric
|
Fluorescence Invitrogen Alexa Fluor 488, 546, 594, 647, and 750 dyes |
Fluorescence
|
| Instrumentation | Fluorescence microscopy or high-content imaging system | High-content imaging system | Fluorescence microscopy or high-content imaging system | Fluorescence microscopy or wide field microscope | Fluorescence microscopy, high-content imaging system, or slide scanner | Flow cytometer |
| Assay format | Coverslips mounted on slides, chamber slides, or 96-well plates | 96- or 384-well plates | Coverslips mounted on slides, chamber slides, or 96-well plates | Tissue sections mounted on microscope slides | Tissue sections mounted on microscope slides
|
Tubes or 96-well v-bottom plates |
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