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Isolate It All: siRNA miRNA ,,, Total RNA Native Protein

RNA Enrichment from Cultured Cells. Total RNA or fractions depleted or enriched in small RNA were isolated in triplicate from 1 x 106 HeLa cells with the mirVana PARIS Kit as per protocol. (A) 1 g of each fraction was resolved on a 1.2% denaturing glyoxal agarose gel and stained with ethidium bromide. (B) 1 g of each fraction was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide. After electrotransfer, miR-16 miRNA was detected by Northern blot with an antisense RNA probe 5' labeled and purified with the mirVana Probe & Marker Kit (Ambion).


Analyze siRNA, mRNA, and Protein Expression in RNAi Experiments
Researchers often need to assess the specificity of gene silencing experiments and to correlate knockdown of a target mRNA with a reduction in the corresponding protein levels. Determining expression levels of active siRNA is also an important parameter that needs to be assessed. Figure 4 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment.

Figure 4. Using the mirVana PARIS Kit to Analyze RNAi Effect. 1.5 x 106 HUVECs were electroporated (800 V, 120 s, 2 pulses, 0.5 s between pulses) with 10 g of siRNA targeting GAPDH mRNA or Silencer Negative Control #1 (Ambion) in 400 l of siPORT siRNA Electroporation Buffer (Ambion) using 4 mm electroporation cuvettes and square wave type pulses. Total RNA and protein were isolated with the mirVana PARIS Kit 48 hours after electoporation. 1 g of total RNA was used to detect the indicated RNA
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