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Got Small RNA?

Total RNA or fractions depleted or enriched in small RNA were isolated from 1x106 HeLa cells with the mirVana PARIS Kit as per protocol. A 1 g aliquot of each fraction was resolved on a 1.2% denaturing glyoxal agarose gel or a 15% denaturing acrylamide gel and stained with ethidium bromide. The same fractions were also analyzed with an RNA 6000 NanoChip on the Agilent 2100 bioanalyzer.


One Sample mRNA +Small RNA + Protein

In addition to efficient isolation of RNA of all sizes and the optional small RNA enrichment procedure, the mirVana PARIS Kit was also designed to quantitatively recover total proteins from the same sample. This is especially important in experiments where the expression level of both the active miRNA or siRNA and the corresponding target protein need to be measured. Figure 3 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment. Here, GAPDH knockdown was triggered by electroporation of a GAPDH-specific siRNA in a primary human cell line (normal human umbilical vein endothelial cells or HUVEC). A reduction of GAPDH expression was observed both at the mRNA and protein levels in these cells, but not in HUVEC cells electroporated with a control Negative Control #1 siRNA sequence (NC). No variation of expression levels was detected for control mRNA (-actin), small RNA (miR-16), and protein (Ku p70).

Figure 3. Using the mirVana PARIS Kit to Analyze RNAi. HUVEC cells (1.5x106) were electroporated with 10
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