( nger (29 nt) than the protected fragmen...)Detecting miRNA & siRNA

nger (29 nt) than the protected fragment generated by the mir-16 probe (22 nt). These probes were then used to simultaneously detect miR-16 miRNA and GAPDH mRNA in total RNA from HeLa cells (Figure 3). The results indicate that both targets are easily detected, and that surprisingly, the level of miR-16 in HeLa cells is greater than that of GAPDH mRNA, a transcript that is known to be highly abundant.

Simultaneous Detection of siRNA Expression and Target Gene Knockdown
For the development of improved siRNA delivery systems and siRNA expression vectors, particularly those that permit targeted delivery or tissue specific expression, researchers need to monitor siRNA levels -- preferably in conjunction with the assessment of target transcript down regulation. To determine if the solution based assay permitted correlation of expressed siRNA levels and targeted mRNA knockdown, probes were generated to a GAPDH siRNA known to effectively reduce GAPDH expression, and to the GAPDH mRNA target. An expression vector (pSilencer 2.0-U6) engineered to express either the GAPDH siRNA or a scrambled control siRNA was transfected into HeLa cells. Three days after transfection, total RNA was isolated and the solution hybridization assay was performed. Using the assay, it was possible to simultaneously detect siRNA expressed from the vector (Figures 3 and 5A), as well as the GAPDH mRNA (Figure 5B). As expected, the GAPDH mRNA levels were found to be reduced considerably (~60%) in cells transfected with the GAPDH siRN
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