( A vector as compared to cell...)Detecting miRNA & siRNA

A vector as compared to cells transfected with the negative control vector (Figure 5B). In addition, the GAPDH siRNA was only detected in the cells transfected with the GAPDH siRNA expressing vector. The specific reduction of GAPDH mRNA expression was similar (~55%) when analyzed by Northern blot.

Figure 5. Analysis of GAPDH siRNA Expression and mRNA Knockdown.(A) HeLa cells were transfected with pSilencer 2.0-U6 engineered to express either an siRNA targeting GAPDH or a negative control siRNA (SCR). Three days after transfection, total RNA was isolated and 1 g was assessed using the mirVana miRNA Detection Kit. Probes to the antisense strand of the GAPDH siRNA were prepared as described in Figure 1. (B) Same experiment with probes specific for GAPDH mRNA or GAPDH siRNA. Both probes had the same specific activity. As a control, GAPDH mRNA expression was also analyzed by Northern blot.

Advantages of the Solution Hybridization Assay
The experiments presented here indicate that the solution hybridization assay based on ribonuclease protection can sensitively and specifically detect small RNAs such as miRNAs and siRNAs. Quantitative analyses can be performed in solution with as little as 10-50 ng of total RNA to detect attomole (10^-18 mol) amounts of target RNA. In general, solution hybridization with short RNA probes is 100500 times more sensitive than membrane hybridization. Another advantage of this assay is the potential to simultaneously detect several small RNAs of the same size or both small RNA
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