by Will Olds
AL
siRNA Knockdown: Implementing Negative Controls in Antibody QC
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 increase in retractions over the past decade.2
and there has been a tenfold 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) knock- down, a powerful negative control method, followed by tips to optimize siRNA transfection experiments.
Using genetic methods to test specificity Because manufacturers and distributors often consider Western blot vali-
dation 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, inhibit- ing 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 consider- ing 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 pre- viously 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 transcrip- tion 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.
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.
AMERICAN LABORATORY 42
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 transfec- tion, 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
JANUARY/FEBRUARY 2017
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