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Detecting miRNA & siRNA

ereas the signal was completely abolished with mir-16 mut probe (Figure 2). This result demonstrates that the assay has the required specificity for detecting small RNAs in total RNA samples.


Detecting Multiple Small RNAs in the Same Sample
Figure 2 also shows that the assay can be used for multi-target detection. The four A residues added to the mir-16 probe between the complementary region and the leader sequence (which is necessary to distinguish protected from unprotected probe) are not cleaved by the mixture of RNases used in the assay. They thus increase the size of the protected fragment by four nucleotides. This probe (mir-16 +4) can be used in the presence of another probe with the same length complementary region but lacking the additional A residues (mir-22) for multi-target detection (Figures 2 and 3). Adding multiple A residues to a small RNA probe sequence permits the detection of more than one small RNA in a single sample giving this assay a distinct advantage over Northern analysis for detecting multiple small RNA molecules that have the same size.

Figure 2. miRNA Expression in Mouse Kidney Total RNA. miR-16 and miR-22 expression was analyzed as in Figure 1 with 1 g of FirstChoice Total RNA from mouse kidney and 32 nt long probes generated with the mirVana miRNA Probe Construction Kit. mir-16 mut probe (32 nt) carries 3 mismatch mutations (ACG to CGA) corresponding to nucleotides 9 to 11 of the miR-16 miRNA sequence. The mir-16+4 probe (36 nt) carries 4 additional A residues between the 22 nt sequence specific for miR-16 and the 10 nt leader sequence, producing a 26 nt lo
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