More and more researchers are conducting siRNA experiments to elucidate cellular pathways and gene function. Often the siRNA effect is only evaluated at the level of mRNA, but this approach overlooks the true subject of most siRNA studies--the biological effect caused by a reduction in the target protein. The biological manifestations of knockdown may not correlate directly with measured mRNA levels. Many proteins remain functionally active long after the corresponding mRNA has been removed. Other proteins need only be present in very small quantities to have a profound biological effect. In these situations, even a significant reduction at the mRNA level may not be effective at producing a cellular response. To fully understand the results of siRNA experiments, analyzing the knockdown of both the target mRNA and the corresponding protein is recommended.
Isolate RNA and Protein from the Same Sample
Until recently, separate samples have typically been required for isolation of high quality RNA and protein. However, when working with rare or difficult-to-obtain samples, it is sometimes impractical to isolate RNA and protein independently. In studies involving large numbers of samples, expensive reagents, or inherent variability (e.g. cell transfection), using separate experimental samples to obtain RNA and protein is not only costly and time consuming, but may also lead to inconsistent results. Ambion's 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.
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 h RNA and protein from your RNAi experiments. Knowing when protein production is reduced will provide information about when cellular phenotypes may arise.
Figure 3. Timecourse of PCNA siRNA Effects on PCNA mRNA and Protein Levels. HeLa cells were plated at 30,000 cells/well into a 24 well culture plate. The cells were transfected with an siRNA against PCNA or with the Silencer Negative Control #1 (scrambled) at a final concentration of 100 nM using siPORT Lipid. Duplicate samples were harvested at 72 hours and each day for 5 days. The PARIS Kit was used to isolate both RNA and protein for either real-time RT-PCR (Panel A) or Western blot analysis (Panel B). RNA was reverse transcribed using the RETROscript Kit; and target cDNA levels were measured by real-time PCR using SYBR Green assays. Input cDNA in the different samples was normalized using real-time data for 18S rRNA. The bar graphs represent an average of three data points. Data are shown relative to mRNA levels of cells transfected with scrambled control siRNA.
Tools to Assist You in Your RNAi Research
Ambion offers products for analysis of both mRNA and protein from the same sample; here we demonstrate the versatility of the PARIS Kit for this application. Other Ambion products used in this study include Silencer Validated siRNAs to Stat-1, p53, and PCNA; and Silencer GAPDH control siRNAs for eliciting RNAi. Also used w ere the RETROscript Kit for cDNA synthesis, and antibodies to GAPDH for analysis of GAPDH at the protein level. Ambion's comprehensive suite of products for analysis of siRNA experiments is appropriate for anyone conducting research involving RNAi.
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Cat# Product Name Size 1710 RETROscript Kit 40 rxns 1921 PARIS Kit 50 purifications 4300 anti-GAPDH, mouse monoclonal 6C5 100 g 4605 Silencer GAPDH siRNA (Human) 5 nmol + 2 nmol Neg Control (50M)