( Figure 1. Sens...)Detecting miRNA & siRNA

Figure 1. Sensitive and Specific Detection of MicroRNAs. (A) The indicated amounts of a 21 nt antisense GAPDH siRNA were spiked into 4 g of yeast RNA and detected with the mirVana miRNA Detection Kit using a 29 nt long probe prepared with the mirVana miRNA Construction Kit. Protected RNA fragments (19 nt) were analyzed on a 15% denaturing polyacrylamide gel. (B) The same experiment as in Panel A with 200 attomoles of sense or antisense GAPDH siRNA radiolabeled probes specific for each strand.

Specificity of the Assay
Any assay for small RNAs must show specificity for the target molecule. Thus, the assay's specificity was investigated. As shown in Figure 1B, radiolabeled probes specific for the antisense strand of GAPDH siRNA detected only the corresponding target RNA. No signal was detected in the absence of target or in the presence of the sense strand of the GAPDH siRNA. Furthermore, the assay yielded no detectable background (e.g. nonspecific hybridization to yeast RNA molecules).

To further investigate assay specificity, miR-16 miRNA was detected in mouse kidney total RNA using three different probes. One probe was perfectly complementary to the target (miR-16), a second included three mismatched nucleotides (mir-16 mut), and a third included four additional A residues between the 22 nt sequence complementary to mir-16 and the leader sequence (mir-16 + 4). The mir-16 and mir-16 + 4 probes were both able to detect miR-16 miRNA in the RNA sample, wh
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