Ultra-sensitive FISH

FISH is a powerful technique that enables the specific detection of target RNA localization in fixed cells. Traditional FISH techniques using large oligonucleotide sequences labeled with one to five fluorophores are generally limited by high background and low sensitivity due to non-specific binding and insufficient signal amplification.

Invitrogen ViewRNA Assays incorporate a proprietary probe set design and branched DNA (bDNA) signal amplification technology. A target-specific probe set of approximately 20 oligonucleotide pairs hybridizes to the target RNA of interest. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, which are formed by Pre-amplifiers, Amplifiers, and fluorochrome-conjugated Label Probes. This methodology results in greater specificity, lower background, and higher signal-to-noise ratios.


Principle of the assay

Target hybridization

A target-specific Probe Set contains 5–40 oligonucleotide pairs that hybridize to specific regions across the target RNA sequence. Subsequent signal amplification steps require that each oligonucleotide pair bind to the target RNA in adjacent positions. Four types of Probe Sets are currently available for RNA detection: Type 1, which is labeled with Invitrogen Alexa Fluor 546 dye; Type 4, which is labeled with Invitrogen Alexa Fluor 488 dye; Type 6, which is labeled with Invitrogen Alexa Fluor 647 dye; and Type 10, which is labeled with Invitrogen Alexa Fluor 750 d ye. When detecting more than one RNA target in a single sample, each Probe Set must be a unique type to differentiate its signal from the others.

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.

Formation of bDNA. A schematic depicting how signal amplification is achieved with bDNA technology.


Select the appropriate assay for your study

 

ViewRNA ISH Cell Assays

ViewRNA Cell Plus Assay

PrimeFlow RNA Assay

mRNA ISH

miRNA ISH

High-content ISH

Cell samples

Cultured cells (adherent or suspension)

Cultured cells (adherent or suspension)

Cultured cells (adherent or suspension)

Cultured cells (adherent or suspension)

Single cell suspensions (primary or cultured)

Multiplexing

Up to four RNA targets (excluding miRNA)

One miRNA and up to two mRNA targets

Up to four RNA targets (excluding miRNA)

Up to three RNA targets (including miRNA)

Up to four RNA targets (including miRNA)

Antibody compatibility

No

No

No

Yes

Yes

Detection signal

Fluorescence

Invitrogen Alexa Fluor 488, 546, 647 and 750 dyes

Fluorescence

Fast Red/AP for miRNA, Invitrogen Alexa Fluor 488 and 750 dyes for mRNA

Fluorescence

Invitrogen Alexa Fluor 488, 546, 647 and 750 dyes

Fluorescence

Invitrogen Alexa Fluor 488, 546 and 647 dyes

Fluorescence

Invitrogen Alexa Fluor 488, 568, 647 and 750 dyes

Instrumentation

Fluorescence microscopy or high-content imaging system

Fluorescence microscopy or high-content imaging system

High-content imaging system

Fluorescence microscopy or high-content imaging system

Flow cytometer

Assay format

Coverslips mounted on slides, chamber slides or 96-well plates

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

Tubes or 96-well v-bottom plates


Search for available (in stock) probe sets

Resources

Support