Ambion, and partner Cenix BioScience, chose qRT-PCR to test Ambion's Silencer Validated siRNAs and to systematically validate more than 1100 siRNAs to verify the effectiveness of Cenix's siRNA design algorithm (this algorithm was used to design all of Ambion's Silencer Validated and Pre-designed siRNAs; see The Easiest Route to Guaranteed Silencing). Figure 1 illustrates the workflow used to validate siRNAs. Using this method, siRNAs that are determined to reduce their target mRNA level by 70% or more are considered "validated" and are subsequently made available as Silencer Validated siRNAs. Figure 2 shows a small subset of validation data generated at Ambion using TaqMan Gene Expression Assays to quantitate target mRNA levels.
←Figure 1. Validation of Ambion's Silencer™ Validated siRNAs. The following procedure is used by both Cenix and Ambion to validate siRNAs:
1) Cells are plated in 96 well plates and grown for 24 hours.
2) Gene specific and negative control siRNAs are independently transfected in triplicate.
3) 48 hours later, RNA is extracted.
4) Target mRNA levels are quantitated by real-time PCR.
5) Data are normalized using 18S rRNA levels.
6) The extent of target gene knockdown is expressed as a percent of mRNA remaining in cells treated with the gene-specific siRNA
compared to cells treated with a negative control siRNA (Silencer Negative Control siRNA #1).
Figure 2. Silencer ™ siRNA Validation Data Generated Using Applied Biosystems TaqMan® Gene Expression Assays. The indicated Silencer Validated siRNAs were transfected into HeLa Cells at 30 nM. RNA was isolated 48 hours later and analyzed by one-step qRT-PCR using the appropriate TaqMan Gene Expression Assay (results were normalized for input RNA amount using real-time data for 18S rRNA). The inset graphs show the reduction in target gene expression compared to cells transfected with an equal concentration of Silencer Negative Control #1. →
Optimizing siRNA Transfection
Before siRNA experiments can be conducted to study gene function, analyze biological pathways, or validate drug targets, optimized siRNA delivery conditions must be identified and/or verified. The best way to test, optimize, and validate siRNA transfection or electroporation conditions is to use a highly effective, verified siRNA to an appropriate target and to monitor decreases in mRNA levels induced by delivery of the siRNA. Ambion uses qRT-PCR as the method of choice, and Applied Biosystems TaqMan Gene Expression Assays provide validated, easy-to-use primer-probe sets for this purpose (Figures 3 and 4).
The Silencer GAPDH siRNA Control--and the TaqMan Gene Expression Assay for GAPDH--provide the ideal control siRNA and assay for optimization of transfection, respectively, (Figure 4). 
←Figure 3. Overview of the Process for siRNA Transfection Optimization.
Figure 4. Using qRT-PCR to Optimize Transfection of siRNA. Cos-7 cells were transfected with CDK2 siRNA or a negative control siRNA using the indicated volumes of transfection agent per well. 48 hr after transfection, the cells were harvested and analyzed by real-time RT-PCR using TaqMan® Gene Expression Assays for CDK2 and 18S rRNA. Data from the 18S rRNA reactions were used to normalize input RNA, and the percent CDK2 expression was calculated as the amount of gene expression compared to the negative control siRNA.↓
Correlating Phenotype with the Extent of Knockdown
To better evaluate functional siRNA experimental results, the phenotype elicited should be correlated with the extent of knockdown induced by a particular siRNA. For a complete picture, both target mRNA levels and corresponding protein levels should be analyzed. Protein levels can be monitored by Western blot, immunofluorescence, ELISA or other means. (Protein can be isolated using Ambion's PARIS™ Kit.) For quantitating mRNA levels, however, qRT-PCR is again the preferred choice.
共3页: 上一页 [1] 2 [3] 下一页
上一篇：RNAi: Background Information 下一篇：cDNA合成
RNAi: Background Informa