Recommended checks and controls for siRNA experiments

Useful information, links and recommended checks and controls to include in your siRNA experiments.

An increasing number of labs are using the siRNA knockdown technique as part of the process to assess the function of a protein within cells. The technique is usually used to determine the effect of removing the protein from the cells:

Does the cell die? Is it a lethal knockout?
Does it affect the expression level of other proteins (particularly within signaling pathways)?
Does it affect the location of other proteins within the cell?
How does it affect cell phenotype, morphology, function?

Brief introduction to siRNA

Small interfering RNA (siRNAs) are 20-25 nucleotide long double-stranded RNA molecules that have a variety of roles in the cell.They are involved in the RNA interference (RNAi) pathway, where they interfere with gene expression by hybridizing to complementary mRNA molecules. This triggers mRNA degradation and suppression of gene expression for a particular gene.

siRNAs were first discovered by David Baulcombe's lab in 1999. In 2001, synthetic siRNAs were shown to induce RNA interference (RNAi) in mammalian cells by Thomas Tuschl in the following paper:

Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494–498. PubMed 11373684.

‘Synthetic’ siRNA oligos designed to hybridize to and degrade target sequences of RNA are now commonly used to induce RNAi in cells. The degradation of the targeted mRNA effectively ‘knocks out’ expression of the corresponding protein. The effects of reduced levels of the target protein can then be analyzed.

How does it work?

Double stranded RNA (dsRNA) is introduced into the cell either using a short oligo siRNA or a DNA plasmid from which a siRNA can be transcribed.
The Dicer protein in the cell digests dsRNA into 21 bp dsRNA (siRNA).
siRNAs are integrated into the RNA Induced Silencing Complex (RISC).
Within this RISC complex, the dsRNAs undergo strand separation. The antisense strand hybridizes to the complementary / target mRNA in the cell.
Nucleases within the activated RISC degrade targeted mRNA.
The fragmented mRNA cannot be translated into protein. This means the protein cannot be expressed, resulting in knockout of the protein.

Optimization of the siRNA sequence for optimal knockout

Many online programs are available to help provide the most suitable siRNA sequence from the mRNA sequence of the protein you wish to knock down.
These computer programs score 21 bp sequences through the full length mRNA of the protein based on the following:

Sequence located within 50-100 nucleotides of the AUG start codon or within 50-100 nucleotides of the termination codon (to ensure transcribed gene is silenced).
siRNA sequence begins with AA (allows use of dTdT at 3’-end of the antisense sequence).This reduces the cost of synthesis and renders the siRNA duplex more resistant to exonuclease activity.
GC content: Ideally the GC content is < 50% (most software defaults range between 40 to 50%)
Stretches of nucleotide repeats.
Avoids sequences with repeats of three or more G’s or C’s
(Initiates intra-molecular secondary structures preventing effective hybridization)
Blast Search (to prevent ‘off targeting’).

Once a target sequence has been chosen, a BLAST search is initiated to ensure that your target sequence is not homologous to other gene sequences.
As a general rule, choose and try three siRNA sequences with the highest score that the program provides, this should give you a high chance that one would work.
Note – this will often be expected by editors for journal publications
Use combinations – start with three SiRNAs and scale down to one
It is possible to knockdown more than two genes at once, but optimize separately first.

Recommended controls and checks for siRNA experiments

The following section provides information on the control samples we would recommend to include in your siRNA experiments. It also includes information on checks that should be carried out when designing and performing the experiment.

Cell line – include one cell line known to have high transfection efficiency.
E.g. 292, HeLa, MRC5, U2OS (We advise not to use primary cells – they do not transfect easily).

An endogenous positive control sample with no siRNA.
As a positive control for the protein of interest and a negative control for siRNA knockout. All reagents other than the siRNA should be added, this checks any effect from the transfection reagents.

Use a dose response curve to optimize the amount of siRNA oligo or plasmid.
To work out an optimal siRNA concentration. There should be enough siRNA to create knockout but at a concentration that does not over-activate the RISC complex or result in toxic effects from other reagents.

Tagged siRNA - observe for example GFP tag fluorescence to confirm transfection.
A small percentage of the siRNA added to the cells can be fluorescently tagged (e.g. GFP) to confirm transfection. Only a small percentage of the total siRNA should be tagged, the rest must be untagged because the tag will prevent RNA binding.

Toxicity controls to check viability of cells.
Calculate and monitor transfection toxicity as some of the reagents can be toxic. This can be done for example by checking cell viability using various cell stains to detect dead cells, e.g. trypan blue.

Induced / non induced.
If the siRNA is being expressed from a plasmid with an inducible promoter, both induced and non induced transfected samples should be tested.

siRNA negative control (using siRNA with a nonsense / scrambled sequence).
siRNA intersects with a number of other pathways, so nonspecific effects can be triggered.
MicroRNAs modulate gene expression largely via incomplete hybridization with a target mRNA and the introduction of an siRNA may cause unintended off-targeting.

Check the sequence of the siRNA (computer programs) – BLAST search.
Blast search the siRNA sequences to ensure the siRNA will hybridize only to the mRNA related to the protein you are interested in.

‘Mock’ control.
Use another protein siRNA e.g. GAPDH (with no target protein siRNA) to check activation of RISC signaling pathway and also that it is not affecting overall cell function.

Check the time for degradation of the mRNA and the existing protein.
The larger the protein, the longer the half life of both the protein and its associated mRNA. You may need to optimize the time required for the siRNA knockdown to take effect on the cells.

Rescue experiment control.
Transfect cells with recombinant protein to re-introduce the protein. Often requested by journals for publication.

Assessing the results – application control samples (e.g. positive and negative controls).
Ensure you include all the required controls for the application you use to assess the results. This will include endogenous positive and negative controls.

Assessing the results:

RT-PCR
To check for presence of the mRNA. Is the targeted mRNA still present? Or has the knockdown been successful?
Very sensitive but doesn’t give an accurate prediction of expected protein levels.

Western blot
Indicates the presence or absence of protein. Can also use antibodies to detect several proteins in the sample and therefore observe the effect the knockout of the target protein has on other proteins.

Immunocytochemistry
Indicates the presence or absence of the knockdown protein.
Advantage – dual staining so can also check effect on expression and cellular location of other proteins.

Troubleshooting summary

How have you selected the sequence? Have you tried more than one siRNA sequence? Check whether the sequence corresponds correctly to the protein.
Have you checked the transfection efficiency and optimized the length of time for knockout to take effect?
Have you checked the transfection by fluorescence tags and the knockout by RT-PCR?
Have you used the correct endogenous positive and negative controls? Have you used scrambled siRNA or standard negative siRNA controls?

Resources

Free online siRNA design and flash tutorial.
National Center for Biotechnology Information provides access to biomedical and genomic information, from where mRNA or cDNA sequence for target selection can be obtained.
Blat tool on UCSC Genome Website.

View our other epigenetic related protocols and techniques.