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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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