QuantiGene® miRNA Singleplex Assay- How It Works
QuantiGene miRNA - Assay Workflow
The QuantiGene miRNA Assay is a hybridization-based assay performed on 96-well plates. The assay is based on DIRECT quantification of the miRNA target using branched DNA (bDNA) signal amplification technology and uses a microplate luminometer for the read out.
Step 1-Sample Preparation: On day one, samples are lysed to release and stabilize miRNA’s. The miRNA assay works with a variety of samples such as: cultured cells, animal tissues, FFPE tissues, whole blood and PAXGene blood, plant tissues, or purified miRNA.
Step 2-Hybridization Step: Overnight hybridization in the 96-well plates with the target specific probe sets (Capture extenders – CE’s and Label Extenders – LE’s).
Step 3-bDNA Signal Amplification: On day 2, Pre-Amplifier (PreAmp) molecules hybridize to each pair of gene specific probe sets, but not to individual probes. Then, multiple Amplifier (Amp) molecules hybridize to each PreAmp. Finally, multiple Label Probe oligonucleotides conjugated to alkaline phosphatase (LP-AP) hybridize to each Amp.
Step 4-Detection: Addition of Chemilumigenic 2.0 Substrate (Lumigen® AP5-5) generates a luminescent signal that is proportional with the amount of target miRNA present in the sample. The signal is read using a luminometer.
Enhanced Detection of miRNA Using Proprietary Chemistry
QuantiGene miRNA probe sets are designed to
- Cover the entire miRNA sequence
- Recognize only mature miRNA and not the longer precorsor miRNA
- Distinguish between closely related family members
Figure 1. micro RNA hybridization with QuantiGene miRNA probe set and bDNA signal amplification.