QuantiGene® miRNA Singleplex Assay- Data & Specifications
micro RNA Probe Sets - Specificity
QuantiGene miRNA probe sets are fully validated for sensitivity and specificity. The probes are tested for family member’s cross-reactivity and are designed to have high specificity towards the target miRNA. The ability of the bDNA miRNA assay to distinguish highly homologous targets with only one base difference was evaluated with synthetic miRNAs of has-let-7 family, miR-18a/b, miR30 family and more. Very low levels of non-specific signal were observed for many miRNAs differing by one or two bases.
QuantiGene miRNA probe sets can differentiate the mature miRNAs from their longer precursors pre-miRNAs. The mature and pre-miRNA probe sets were designed and assayed with mature and pre- miRNA target molecules. The mature miRNA probe sets only showed ~5% signal with pre-miRNA targets compared to mature miRNA molecules using 600,000 and 6,000,000 copies. This indicated that mature miRNA assay is highly specific to mature miRNA while pre-miRNA probeset design shows high specificity for pre-miRNA (Figure 2).
Figure 2. micro RNA probe sets specificity - discrimination between mature and precursor miRNA targets.
miRNA Profiling in a Variety of Samples
QuantiGene miRNA Assay offers excellent spike recovery for miRNA with a ±20% recovery rate. The spike recovery rates are excellent across a variety of sample types: cells lysates, corn husk lysate, liver tissue lysate, stomach tissue lysate, blood lysate and PAXgene blood lysate (Figure 3). The assay linearity and spike recovery were tested using a 23 bp unique synthetic miRNA sequence that is not present in any species.
Figure 3. Excellent miRNA quantitation in a variety of samples using direct from lysate analysis.
miRNA Up-regulation Upon Differentiation
Mouse C2C12 differentiated and undifferentiated cells were lysed and used to analyze known miRNA’s that are overexpressed during differentiation. miR-1, miR-133a, miR-206 and myogenin (myogenin was analyzed using QuantiGene 2.0 RNA Assay) were analyzed (Figure 4) and the data obtained is in concordance with published data.
Figure 4. miRNA up-regulation inC2C12 mouse cells upon differentiation.
Accurate Determination of miRNA Copies in Cancer Tissue
QuantiGene 2.0 miRNA Assay was used to analyze expression levels of miR-155 in human breast normal and tumor tissues and provided accurate results. The miR-155 expression in normal tissue was ~488,000 copies and in the cancer tissue was ~4,900,000 copies.
Figure 5. miRNA expression analysis in cancer and normal tissue sections.
Concordance to GeneChip® miRNA Array 2.0
QuantiGene miRNA Assay can be used to validate miRNA array results. To evaluate the concordance of fold changes between QuantiGene miRNA assays and GeneChip miRNA Array data, we performed regression analysis of fold differences in Ambion FirstChoice Human Brain Total RNA (AM7962) and Ambion FirstChoice Human Lung Total RNA (AM7968) using QuantiGene data of 20 miRNAs and GeneChip miRNA Array data (Figure 6). The R2 between these two assays was 0.74, indicating a good fold-change correlation.
Figure 6. QuantiGene miRNA Assay and GeneChip miRNA Array data concordance. 200ng human brain and lung total RNAs were labeled with flash tag HSR in duplicate and hybridized to Affymetrix GeneChip miRNA Arrays. Replicate Log2 signal intensities were averaged. To determine Brain/Lung Fold Change, lung average Log2 signal was subtracted from brain average Log2 signal.
- Rayner K. J., et al. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature 478(7369):404-7 (2011).