( Figure 2. Effects ...)RNAi: Get the Whole Story

Figure 2. Effects of Stat-1 and p53 siRNAs on Target mRNA and Protein Levels. HeLa cells and 293T cells were plated at 30,000 cells/well into 24 well culture plates. The cells were transfected using siPORT Lipid with an siRNA targeting p53 (293T) or Stat-1 (HeLa) or with a scrambled negative control siRNA (Silencer Negative Control #1) at a final concentration of 100 nM. Samples were harvested 72 hours after transfection and were subjected to RNA and protein isolation using the PARIS Kit. RNA was reverse transcribed using the RETROscript Kit, and target cDNA levels were analyzed by real-time PCR using SYBR Green detection. Target gene expression in the transfected cells was compared to cells transfected with an equal concentration of the Silencer Negative Control #1 siRNA. Input cDNA in the different samples was normalized using real-time PCR data for 18S rRNA. The bar graphs represent an average of three data points.

Timecourses: The Best Way to Understand siRNA Effects
To get the best picture of the biological effects of an siRNA on its target, we recommend conducting a time course experiment to monitor both RNA and protein levels over time. In Figure 3 a time course experiment was performed after the introduction of an siRNA against PCNA (Proliferating Cell Nuclear Antigen). Total RNA and protein were isolated from the same sample at days 3, 4, and 5 and analyzed by either real-time RT-PCR or Western blot. The maximal reduction of message for PCNA was achieved by day 3; however, the maximal reduction of protein did not occur until day 4. These data emphasize the utility of the PARIS Kit for analysis of bot
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