RNA interference
A chemically synthesised 21-nucleotide duplex with 2-nucleotide 3′ overhangs, delivered into the cytoplasm by cationic lipid, electroporation or a conjugate. It is loaded straight into Argonaute without Dicer, gives the deepest and fastest knockdown of the three formats, and fades as the cells divide: deepest at 24–48 h, usually gone within a week.
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siRNA
A chemically synthesised 21-nucleotide duplex with 2-nucleotide 3′ overhangs, delivered into the cytoplasm by cationic lipid, electroporation or a conjugate. It is loaded straight into Argonaute without Dicer, gives the deepest and fastest knockdown of the three formats, and fades as the cells divide: deepest at 24–48 h, usually gone within a week.
How it differs from the standard method
- The whole dose arrives at once, so both silencing and seed-driven off-target repression peak on the first day.
- Nothing is integrated, so the same cells can be used for a rescue or a second duplex, and there is no selection step.
- Delivery is the limiting factor: hard-to-transfect lines, primary cells and suspension cultures often need electroporation instead of lipid.
- A duplex can be chemically modified (2′-O-methyl in the seed region, for instance) to lower off-target repression without changing the target site.
- Repeat transfection at 48 h is the usual way to hold a knockdown for a four- or five-day assay.
1Borrowing a pathway the cell already has
RNA interference is not a technique someone invented so much as a pathway someone learned to feed. Animal cells already use short double-stranded RNA to regulate their own messages: a duplex is handed to Argonaute, one strand is kept as the guide, and the resulting complex scans the cytoplasm for messages the guide can pair with.
2How much, and for how long
Because the question is quantitative, the answer has to be measured rather than assumed. A good duplex at 10–25 nM typically removes 70–90% of its target message within 24 hours, and the message stays low while the duplex lasts.
siRNA
A synthetic 21-mer duplex delivered by lipid, electroporation or conjugate. Deepest knockdown, no genomic change, gone within a week. The whole dose arrives at once, so the seed concentration is at its highest on the first day.
shRNA
A hairpin transcribed from an integrated vector and processed by Dicer. Holds for weeks and survives selection, at the cost of lentiviral work, copy-number variation and competition with the cell's own microRNAs.
esiRNA
A long dsRNA against one transcript, diced in vitro into a heterogeneous mixture. No single seed reaches a concentration where its off-targets matter, which is the point; batch-to-batch composition is the price.
Independent sequences
Two or three duplexes against different sites in the same transcript share the target and nothing else. A phenotype all of them produce, in proportion to how deeply each one knocks the gene down, is the gene's.
3What a knockdown experiment has to control for
Three artefacts account for most retracted knockdown phenotypes, and each has a control that catches it. Seed-driven off-target repression is caught by independent sequences, or by rescuing the phenotype with a construct the duplex cannot target.
- RISC
- RNA-induced silencing complex: Argonaute loaded with a single guide strand, plus its associated proteins. The functional unit that finds and cleaves the message.
- Guide and passenger strand
- The two strands of the duplex. Thermodynamic asymmetry decides which is kept; a badly designed duplex loads the wrong strand and silences whatever that strand matches.
- Seed region
- Positions 2–8 of the guide. Pairing here alone is enough for microRNA-like repression, and it is the origin of most off-target effects.
- Non-targeting control
- A duplex with no predicted target in the genome, used at the same concentration as the active one. It controls for delivery, for loading RISC and for the interferon response.
- Rescue
- Re-expressing the target from a construct that lacks the duplex's binding site. If the phenotype disappears, it was on-target.
- Knockdown versus knockout
- RNAi lowers the level of a message; a nuclease removes the gene. Partial loss can give a different phenotype from total loss, and sometimes a more informative one for an essential gene.
- Interferon-stimulated gene
- A gene such as IFIT1 or OAS1 induced when the cell senses foreign RNA. Two assays for these say whether the innate response contaminated your experiment.
- Exportin-5 saturation
- Highly expressed hairpins compete with endogenous pre-microRNAs for nuclear export and for Argonaute, so a strong shRNA can disturb the whole microRNA system.
4How it fails
- Shallow knockdown with no phenotype. Usually delivery: a confluent monolayer takes up very little lipoplex, complexes formed in serum barely form at all, and some lines simply do not transfect. Check with a positive-control duplex against an abundant housekeeping message before concluding the gene does not matter.
- Deep mRNA knockdown with an unchanged blot. The protein has not turned over yet. Harvest later, re-transfect at 48 hours, or accept that the protein is too stable for this method and consider a degron or a knockout.
- A phenotype one sequence gives and another does not. Most often a seed effect, sometimes one sequence being far more potent than the other. Plot phenotype against measured knockdown across all the sequences before deciding which it is.
- Everything in the well looks sick, including the control. Lipid toxicity, an interferon response, or both. Titrate the reagent against a fixed duplex concentration and read the control wells first.
- A stable line whose knockdown has faded. Populations are selected for the cells that express the hairpin least, and the cassette can be silenced. Re-measure the knockdown at every harvest and keep the passage number low.
Key terms
Practise these as flashcards →A synthetic double-stranded RNA, usually 21 nucleotides with 2-nucleotide 3′ overhangs, that enters RISC directly without needing Dicer.
A short hairpin RNA transcribed in the cell from a plasmid or integrated vector, processed to a duplex before loading. The way to make a knockdown last.
Repression of transcripts other than the intended one, mostly through seed complementarity in their 3′ UTRs. Scales with guide concentration.
The fraction of the target removed, relative to a non-targeting control measured in the same experiment. Quote it with the method and the time point, because it is a property of both.
The fraction of cells that take up the reagent. Low efficiency looks exactly like a weak duplex in a population measurement.
Multiplicity of infection for a viral shRNA: functional particles per cell. Transduction is Poisson, so MOI sets both the fraction transduced and the copies per cell.
The RNase III enzyme that cuts long double-stranded RNA and shRNA hairpins into ~21-nucleotide duplexes. The step a synthetic siRNA skips.
A target coding sequence altered or truncated so the duplex cannot bind it, expressed to test whether a phenotype is on-target.
When to use it
- Asking what a gene does when you need the answer in days rather than weeks, and in a cell line where a knockout would be lethal or slow to make.
- Studying an essential gene, where partial loss is informative and complete loss only kills the cells.
- Testing many genes in parallel, an arrayed or pooled screen, where making a knockout for each one is out of the question.
- Depleting a protein in a cell type that cannot be clonally selected, such as primary cells or differentiated cultures, where a transient duplex is the only practical route.
- Checking a hypothesis quickly before investing in a knockout line, with the understanding that the knockdown phenotype will need the knockout, or a rescue, to stand up.
Compared with alternatives
A different mechanism for the same goal: RNase H cuts the RNA in a DNA/RNA duplex, which works in the nucleus. Prefer it for nuclear-retained transcripts, for redirecting splicing, and where free uptake without a transfection reagent matters.
Removes the gene instead of lowering the message, so loss is complete and permanent. Prefer it when you need a clean null, and prefer RNAi when the gene is essential or you need the result this week.
The pooled, genome-scale version of the same question. Prefer it for discovery; RNAi screens remain useful where partial loss of function is the phenotype of interest.
The measurement that decides whether a knockdown is real. Prefer reading the protein over the message whenever an antibody exists, because the phenotype can only be caused by the protein.
How knockdown of the message is quantified, against a non-targeting control and a reference gene. Fast and sensitive, but it measures the reaction rather than the loss of function.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A chemically synthesised 21-nucleotide duplex with 2-nucleotide 3′ overhangs, delivered into the cytoplasm by cationic lipid, electroporation or a conjugate. It is loaded straight into Argonaute without Dicer, gives the deepest and fastest knockdown of the three formats, and fades as the cells divide: deepest at 24–48 h, usually gone within a week.
- The whole dose arrives at once, so both silencing and seed-driven off-target repression peak on the first day.
- Nothing is integrated, so the same cells can be used for a rescue or a second duplex, and there is no selection step.
- Delivery is the limiting factor: hard-to-transfect lines, primary cells and suspension cultures often need electroporation instead of lipid.
- A duplex can be chemically modified (2′-O-methyl in the seed region, for instance) to lower off-target repression without changing the target site.
- Repeat transfection at 48 h is the usual way to hold a knockdown for a four- or five-day assay.