ViewRNA High Content Screening Assay- How It Works
ViewRNA HC Screening Assay—Assay Workflow
|Click on image to enlarge|
Sample Preparation. Adherent or suspension cells are fixed and permeabilized.
Target Hybridization. Gene-specific Probe Sets hybridize to target mRNAs. For simplicity, a three-plex assay with only single oligonucleotide pairs is shown. A typical Probe Set contains 20 or more oligonucleotide pairs.
Signal Amplification. Independent but compatible signal amplification systems enable simultaneous detection of multiple RNAs in a single assay. Distinct sets of Pre-Amplifier (PreAMP), Amplifier (Amp) and Label Probe (LP) molecules are used to detect different target mRNAs. A fully assembled signal amplification “tree” has 400 binding sites for each Label Probe. When all target-specific oligos in the Probe Set bind to the target mRNA transcript, an 8,000 fold amplification occurs for that one transcript.
Detection. Target mRNAs are visualized using a standard fluorescence microscope or automated imaging platform with the corresponding filter sets (EX: 488, 550, 650 and 740 nm).
Selecting the Target Specific Probe Sets
IMPORTANT: TYPE 4 Signal Amplification ONLY works with TYPE 4 Target Specific Probe Set. TYPE 4 Label Probes ONLY work with TYPE 4 Signal Amplification. When multiplexing for each different target choose a different signal amplification type and a different dye for the LP-Dye Probe. Read More ...
Compatible Sample Types
The ViewRNA HC Screening Assay has been optimized for analyzing suspension or adherent cultured cells. However other samples may be used - like circulating tumor cells, cells isolated from blood or other body fluids. We do not provide a protocol for the analysis of target RNAs from those starting materials. However we do recommend that you follow the protocol for analyzing suspension cells and you optimize as necessary. This assay has been optimized for automation. If you would like to run your samples in manual mode please use the ViewRNATM ISH Cell Assay.
Specificity of the Target Specific Probe Sets
To demonstrate the target specific probe sets specificity we utilized 18S rRNA as a model and used the ViewRNA ISH Cell Assay. 18S rRNA signal was amplified using a standard 16X amplifier (a). The target specific probe set is comprised of twenty oligonucleotide pairs. In order for a signal amplification branch to bind to the target mRNA, both oligos from the oligonucleotide pair need to hybridize side by side to the target mRNA. Read More ...
Single Transcript Sensitivity
To demonstrate single transcript sensitivity, therefore one dot is equivalent to one target molecule, we utilized a RNA model system. In the RNA model system, two Probe Sets were designed to target different regions of the ERBB2 (Her-2) target. One Probe Set targeted the region from exons 2-9 and was compatible with the signal amplification system using a fluorescent molecule with excitation at 550nm and the other Probe Set targeted the region from exons 10-20 and was compatible with the signal amplification system using a fluorescent molecule with excitation at 488 nm. Read More ...