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cells transcribe the foreign DNA to generate the shRNA described above (Figure 2). Afterward, the enzyme Dicer processes the shRNA into siRNA by removing the loop sequence. The subsequent siRNA binds with RISC (RNA-induced silencing complex), which divides the two strands of the RNA and activates the complex. RISC remains bound to one strand that complementarily binds to a target mRNA and degrades it, resulting in diminished production of the associated protein.


5. Validation of siRNA data


One way to validate the siRNA data is to titrate the siRNA. Consult the manufacturer’s instructions, which generally recommend a concentration of 5–100 nM, and use the lowest working concentration to ensure target specificity. Another way to validate data is to monitor RNA and protein levels throughout the experiment. Successful mRNA silencing without a simultaneous decrease in protein levels suggests a slow protein turnover. In general, the earliest time after which the silencing effect can be ob- served is 24 hours.


6. Test and evaluate


In a successful knockdown antibody validation experiment, the gene is efficiently downregulated and the signal is diminished in the knockdown sample compared to the controls in a Western blot. If bands are non- specific or inconsistent across all of the Western blot membrane, there may be an error in the experiment or the antibody may be nonspecific. At this point, reevaluate the protocol and experimental design to ensure the conclusion is valid.


Conclusion


Although negative controls in antibody validation are vital to providing gold-standard products, the amount of time and resources validation


protocols require of individual labs is demanding. With an appropriate amount of data and reassurance from previous published studies that mention the reagent catalog number and manufacturer, Western blot validation with only positive controls is a second-best alternative. The scientific community is urged to aspire to higher antibody validation standards and incorporate negative controls such as siRNA knockdown in its processes. From these unified efforts, we will make reproducible research commonplace, improve the quality of research and accelerate scientific progress.


References


1. Baker, M. Antibody anarchy: a call to order. Nature 2015, 527(7579), 545–51.


2. http://www.the-scientist.com/?articles.view/articleNo/45134/title/ Antibody-Alternatives/


3. Wittrup, A. and Lieberman, J. Knocking down disease: a progress report on siRNA therapeutics. Nature Rev. Genetics 2015; 16(9), 543–52.


Will Olds, Ph.D., is scientific officer, Proteintech, 5400 Pearl St., Ste. 300, Rosemont, Ill. 60018, U.S.A.; tel.: 888-478-4522; e-mail: will@ptglab.com; www.ptglab.com


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Figure 2 – Successful transfection results in cells transcribing the foreign DNA to generate the shRNA, leading to diminished production of the target protein.


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