QuantiGene miRNA Singleplex Assay: How It Works

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 readout.

Step 1. Sample preparation: On day one, samples are lysed to release and stabilize miRNAs. 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.

QuantiGene miRNA probe sets are designed to:

• Cover the entire miRNA sequence 
• Recognize only mature miRNA and not the longer precursor miRNA 
• Distinguish between closely related family members

Figure 1. microRNA hybridization with QuantiGene miRNA probe set and bDNA signal amplification.