siRNA Knockdown: Implementing Negative Controls in Antibody QC

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Negative controls are an essential part of experimental design that has been overlooked by antibody manufacturers and distributors. Without routine negative controls, nonspecific, poorly validated antibodies have become a major contributor to the reproducibility crisis in biomedical research. The scientific community spends an estimated $800 million each year purchasing low-quality antibodies,¹ and there has been a tenfold increase in retractions over the past decade.² Without new, thorough and more standardized validation methods, these problems will continue to impair research and scientific progress.

This article presents an overview of short interfering RNA (siRNA) knockdown, a powerful negative control method, followed by tips to optimize siRNA transfection experiments.

Using genetic methods to test specificity

Figure 1 – Combination of siRNA-treated cells and a specific antibody results in a drop in signal compared to an untreated sample by Western blot.

Because manufacturers and distributors often consider Western blot validation complete after a positive result, they rarely test whether antibodies still produce a signal when the target protein is suppressed or removed. One efficient method for diminishing protein levels is siRNA knockdown. This method degrades messenger RNA (mRNA) after transcription, inhibiting translation of the target protein.³ Compared to an untreated sample by Western blot (Figure 1), the combination of siRNA-treated cells and a specific antibody will result in a substantial drop in signal.

siRNA knockdown is an intricate process. The following guidelines, based on Proteintech (Rosemont, Ill.) methods, are offered for anyone considering how to validate antibody reagents or seeking a greater understanding of the siRNA validation procedure in general. Those working with a new target cell line should be prepared to run multiple test transfections to optimize conditions.

1. Create an RNase-free environment

Prior to starting an experiment, the work space should be cleaned with an RNase-decontaminating solution. Pipettes with RNase-free tips are best and should be stored carefully to avoid cross-contamination. Always use gloves when working with siRNA, changing them after touching any surface.

2. Include essential siRNA controls in the experimental design

The knockdown experiment must include three parts: 1) a condition in which the siRNA is targeted toward the gene of interest, 2) a condition in which a “scramble” siRNA is used to control for nonspecific changes in gene expression and 3) the normal gene expression level should be determined by incorporating a nontransfected control. The experiment is even stronger if a second siRNA is used against the same target but another region of the mRNA and similar results are obtained. Fluorescent labeling of the siRNA simplifies targeting of the knockdown effect.

3. Designing siRNA against the gene

Once the siRNA target site is chosen, an appropriate vector needs to be designed (many online resources are available). When possible, use a previously published siRNA sequence to optimize knockdown of the target of interest as a positive control. Aside from the actual engineering of the vector, this step is relatively simple.

After designing the short hairpin RNA (shRNA) that is the precursor form of the siRNA, the ultimate aim is to design two single-stranded 19–22 mer DNA oligonucleotides: one sense strand and one antisense strand. Proteintech recommends 21–23 nt sequences and a guanine-cytosine (GC) content between 30 and 50% to be stable enough to form the hairpin but not so stable that it cannot be made into single-stranded RNA. Their transcription products will eventually recombine, linked at one end by a short loop sequence, such as TTCAAGACG. There should be no sequence that shows homology to other coding sequences, and the siRNA should not bind to introns.

4. Transfection and cell culture

When vector production is complete, a suitable transfection method should be determined for introduction into cells. At the time of transfection, cells should be in optimal physiological condition and be passaged frequently. A cell density of around 70% is needed. Be sure culture conditions remain constant throughout. If transfection is successful, the 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.

 Figure 2 – Successful transfection results in cells transcribing the foreign DNA to generate the shRNA, leading to diminished production of the target 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 observed 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 nonspecific 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: [email protected]; www.ptglab.com