miR-ID® microRNA Detection Platform

miR-ID® is a novel platform for detecting miRNA using a circularization-based RT-qPCR method. miR-ID® is highly sensitive, uses single-dye detection, and can discriminate miRNA isoforms with single nucleotide differences at any position along the molecule. The technology works well with all sample sources, including total RNA, cell lysates, and tissue lysates.

Figure 1: Schematic of miR-ID®. Step 1: Circularization of a linear miRNA; Step 2: Reverse transcription mediated by rolling circle amplification to obtain multimeric cDNA; Step3: Quantification of the amplicon by RT-qPCR using highly sequence-specific 5’-overlapping primers and SYBR Green detection.

miR-ID® provides better sensitivity, specificity, and versatility than competing methods. In addition, miR-ID® is the only PCR-based platform that is able to distinguish small RNAs with commonly occurring end modifications such as 5' triphosphates (e.g., secondary siRNAs in C. elegans), and 2'-OMe modifications (e.g., 3' ends of piRNAs in animals and all small RNAs in plants). Such end modifications are thought to play a role in both the stability and function of small RNAs, and there is a clear need to differentiate and detect modified small RNAs from their unmodified versions.

Comparison of miR-ID® and the leading competitor for small RNA detection

	miR-ID®	Leading Competitor Assays
Sensitivity	++++	++++
Specificity	++++	++
Sequence coverage	++++	+++
Distinguish and quantify RNA end modifications	Yes	No
Need for modified probes	No	Yes
Cost	$	$$
Discrimination between precursor and mature miRNAs	+++	+++
Works in cell lysates	Yes	Yes
Multiplex capability at RT step	Yes	Yes
Restrictions on primer position	None	Restricted to miRNA ends

Sensitivity

The miR-ID® miRNA detection platform provides a higher signal-to-noise ratio than leading competitors, while using unmodified DNA primers, single-dye detection (SYBR green or EVA green), and no specialized probes. These features help keep the costs low and allow for a rapid development of assays for miRNAs of interest.

Figure 2: Standard curve for lin-4 by miR-ID® detection by qPCR using miR-ID® and a probe-based competitor assay.(click image to enlarge)

Specificity

miR-ID® provides unmatched discrimination between miRNA isoforms with single nucleotide differences. There is virtually no cross-talk between miRNAs from the same family, as well as superior discrimination between pre- and mature miRNAs.

Figure 3a: miR-ID® (left panel) provides exceptional discrimination of miRNAs with nucleotide polymorphisms at the 5’ end as opposed to a probe-based competitor assay (right panel). (click image to enlarge)

Figure 3b: miR-ID® (left panels) provides exceptional discrimination of miRNAs with nucleotide polymorphisms at the center as opposed to a probe-based competitor assay (right panels). (click image to enlarge)

Figure 3c: miR-ID® provides exceptional discrimination of closely related miRNAs of the miR-17-92 cluster. (click image to enlarge)

Quantification of End-Modified Small RNAs

miR-ID® is the first platform that can quantitatively discriminate between small RNAs with an identical sequence but differing terminal modifications. This is particularly useful in plant miRNAs, where discrimination between 2-O-methyl modified and unmodified miRNA is of considerable interest.

Figure 4: miR-ID® can quantitatively detect 2' O-methyl modifications at the 3’-terminus of miRNAs. By selecting enzymes that circularize either both types of miRNAs (with 2’-OMe or 2’-OH forms on the 3’termini), shown in green, or 2’-OH forms only (shown in red), these two end-modifications can be distinguished. In this example, synthetic let-7b miRNAs with the two respective 3’ termini were assayed and quantification with the enzyme circularizing both forms were set to 100%. (click image to enlarge)

Convenience

miR-ID® can accommodate a variety of RNA sources without purification, including tissue or cell lysates. For the analysis of total RNA, no enrichment for small RNAs is required: miR-ID® works as well with total RNA as with enriched fractions.