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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:
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.
The siRNA can be introduced into the cell by either:
siRNA delivery options
siRNA can be directly introduced into the cells by using microinjection or electroporation. Another popular option, most often used while working with cell cultures, is using a transfection reagent (calcium phosphate, liposome-based reagent). On the market there are several specialized siRNA-delivery reagents from various vendors. Usually transient RNAi effect is seen within 4 hours and the maximum down regulation can be observed in 24-47 hrs. The effect lasts several cell generations and from 4-10 days depending on cell culture type.
Characteristics of the siRNA
siRNA-transcribing dsDNA introduced into the cell is typically ~21 bp with 3’ nucleotide overhangs.
A small hairpin loop can be included for stability. This is siRNA which is often introduced into the cell by transfecting a plasmid. The hairpin is enzymatically cleaved to leave double stranded siRNA.
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:
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.
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.
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.
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.
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.
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.