( a variety of sample types and ...)Detecting miRNA & siRNA

Solution Hybridization Improves Sensitivity
Because hybridization of probe to target in solution is more sensitive than hybridization on solid support (Northern analysis), Ambion investigated the solution based RNase protection assay for its ability to detect small RNAs. After modification of the hybridization conditions to maximize hybridization of short probes to their targets, the assay's ability to specifically detect small RNA molecules were analyzed.

To examine the sensitivity of the assay, decreasing amounts of a 21 nt RNA, corresponding to the antisense strand of an siRNA known to target GAPDH, were spiked into a yeast RNA sample and then analyzed by the solution hybridization method. In brief, the samples were mixed with a 29 nt high specific activity radiolabeled probe (5 x 10⁴ cpm) and hybridization buffer. After heat denaturation, each mixture was incubated at 42C to hybridize the probe to the complementary siRNA target strand. Unhybridized RNA species and excess RNA probe were removed by a brief ribonuclease digestion. Protected target RNA fragments were recovered in the same tube using a reagent that simultaneously inactivates the ribonuclease and precipitates the nucleic acid. The protected RNA was then resuspended with gel loading buffer, analyzed on a denaturing polyacrylamide gel, and exposed to film. Figure 1A demonstrates that the assay was able to detect as little as 50 attomoles of the 21 nt target RNA after a two hour exposure.
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