( /catalog/CatNum.php?1921PARIS ...)RNAi: Get the Whole Story

/catalog/CatNum.php?1921">PARIS Kit was developed specifically to address these problems. It allows researchers to isolate both RNA and protein from a single experimental sample. Figure 1 shows both Northern and Western data from an siRNA experiment in which cells were transfected with either a chemically synthesized siRNA or with a plasmid expressing a hairpin siRNA--both targeting human GAPDH. The PARIS Kit was used to isolate RNA and protein from either total cell lysates, or from the nuclear or cytoplasmic cell fraction. The cellular localization of GAPDH in the cytoplasm is reflected in this data, which shows detection of GAPDH mRNA and protein only in the cytoplasmic cell fraction.

Figure 1. Effects of GAPDH siRNA on GAPDH mRNA and Protein Levels. HeLa cells were plated at 200,000 cells/well into a 6 well culture plate. 24 hours later they were transfected with either a chemically synthesized siRNA at a final concentration of 100 nM or with pSilencer 2.0-GAPDH; both target human GAPDH. Samples were harvested 48 hours after transfection, and both RNA and protein were isolated using the PARIS Kit. mRNA knockdown was evaluated by Northern blot using an antisense radiolabeled RNA probe transcribed from pTRI-GAPDH human (Ambion Cat #7430). For the Western blot, anti-GAPDH antibody (Ambion Cat #4300) was used.

Figure 2 also presents evidence at both the mRNA and protein level for knockdown of two different genes, Stat-1 and p53. In these experiments, cells were harvested for analysis 72 hours after transfection. These results are typical in that a significant RNAi effect can be seen 72 hours post-transfection.

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