RNA FISH

Ultra-sensitive RNA FISH

RNA FISH (RNA fluorescence in situ hybridization) is a powerful technique that enables the visualization and localization of RNA and protein targets in fixed cells. Ultra sensitive RNA FISH using Invitrogen ViewRNA and PrimeFlow RNA Assays incorporate a proprietary probe set design and branched DNA (bDNA) signal amplification technology, which measure RNA directly from the sample source, without RNA purification or enzymatic manipulation. RNA FISH using bDNA technology results in greater specificity, lower background, and higher signal-to-noise ratios.

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 cytomety. 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.

RNA FISH using branched DNA technology

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.

RNA FISH signal amplification

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 ViewRNA Tissue 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 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 miRNA)
Antibody compatibility No No Yes No Yes
Detection signal

Fluorescence


Invitrogen Alexa Fluor 488, 546, 647, and 750 dyes

Fluorescence


Invitrogen Alexa Fluor 488, 546, 647, and 750 dyes

Fluorescence


Invitrogen Alexa Fluor 488, 546, 647, and 750 dyes

Fluorescence or colorimetric


Fast-red and Fast Blue

Fluorescence


Invitrogen Alexa Fluor 488, 568, 647, and 750 dyes

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 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 Tubes or 96-well v-bottom plates

Search for available (in stock) probe sets

Need to detect RNA in a plate reader format?
See QuantiGene SinglePlex assays for RNA detection

Resources

Support