The QuantiGene Plex Gene Expression Assay is a hybridization-based assay using the xMAP™ Luminex beads and performed on 96 or 384-well plates. The assay is based on DIRECT quantification of the RNA targets for multiplexing of 3 to 80 RNA targets and branched DNA (bDNA) signal amplification technology. On the first day the sample is lysed to release the RNAs, and incubated overnight with target specific probe sets. On the second day the signal amplification tree is built via sequential hybridization of PreAmplifier, Amplifier, and Label Probe. Each amplification unit gives a 400x signal amplification and there are six amplification units per target RNA copy resulting in a 2,400x signal amplification per copy RNA. The signal is detected by adding SAPE substrate and using a Luminex instrument for results and analysis.

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Sample preparation

Step 1. Sample preparation: Samples are lysed to release and stabilize RNAs. The RNA assay works with a variety of samples such as: cultured cells, human, plant and animal tissues, FFPE tissues, whole blood and PAXGene blood, or purified RNA.

Target hybridization

Step 2. Target hybridization: Overnight hybridization in the 96 or 384-well plates with the target specific probe sets panel (Capture extenders – CE’s, Label Extenders – LE’s and blocking probes).

Signal amplification

Step 3. Signal amplification: Signal amplification is achieved using branch DNA (bDNA) technology. A Pre-Amplifier molecule hybridizes to each pair of Label Extenders, but not to individual probes. Then, multiple Amplifier molecules hybridize to each PreAmp lifier. Finally, multiple Label Probe oligonucleotides hybridize to each Amp lifier.

Detection

Step 4. Detection: Addition of streptavidin phycoerythrin (SAPE) generates a signal that is proportional with the amount of target RNA present in the sample. The signal is read using a Luminex instrument.

Sample preparation

Step 1. Sample preparation: Samples are lysed to release and stabilize RNAs. The RNA assay works with a variety of samples such as: cultured cells, human, plant and animal tissues, FFPE tissues, whole blood and PAXGene blood, or purified RNA.

Target hybridization

Step 2. Target hybridization: Overnight hybridization in the 96 or 384-well plates with the target specific probe sets panel (Capture extenders – CE’s, Label Extenders – LE’s and blocking probes).

Signal amplification

Step 3. Signal amplification: Signal amplification is achieved using branch DNA (bDNA) technology. A Pre-Amplifier molecule hybridizes to each pair of Label Extenders, but not to individual probes. Then, multiple Amplifier molecules hybridize to each PreAmp lifier. Finally, multiple Label Probe oligonucleotides hybridize to each Amp lifier.

Detection

Step 4. Detection: Addition of streptavidin phycoerythrin (SAPE) generates a signal that is proportional with the amount of target RNA present in the sample. The signal is read using a Luminex instrument.

Video: Invitrogen QuantiGene Plex Assay-How it Works