Many of our customers are now 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 signalling pathways)?
- Does it affect the location of other proteins within the cell?
- How does it affect cell phenotype, morphology, function?
- Brief introduction to siRNA
- How does it work?
- Optimization of the siRNA sequence for optimal knockout.
- Recommended controls and checks for siRNA experiments.
- Assessing the results
- Troubleshooting summary
Small interfering RNA (siRNA) 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 it interferes with the expression of a specific gene by hybridizing to its corresonding RNA sequence in the target mRNA. This then activates the degrading mRNA. Once the target mRNA is degraded, the mRNA cannot be translated into protein.
SiRNAs were first discovered by David Baulcombe's. 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): 494–8. PubMed 11373684.
‘Synthetic’ siRNA oligos designed to hybridize to and degrade target sequences of RNA are now commonly used to induce RNA interference 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 analysed.
For further information on siRNA:
- ds RNA is introduced into the cell either using a short oligo (Short Interfering RNA) siRNA or a DNA plasmid from which an siRNA can be transcribed.
- The Dicer protein in the cell digests dsRNA into 21 bp ds RNA (siRNA).
- siRNA’s are integrated into the RNA Induced Silencing Complex (RISC).
- Within this RISC complex, the double stranded siRNA’s 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.
The siRNA can be introduced into the cell by either:
- siRNA oligo
Advantage: Fast, easy, effective transient siRNA knockdown.
Disadvantage: Transient effect (not stable)
- DNA based or retroviral RNA based Plasmid / Vector
Advantage: Regulated (inducible) knockout for functional/recovery/lethal knock
Can use reporter/selection genes
Transient or stable knockout (however, cells usually won’t survive long)
Characteristics of the siRNA
siRNA ds DNA introduced into the cell is typically approximately 21 bp with 3’ nucleotide overhangs.
A small hairpin loop can be included for stability. This is shRNA which is often introduced into the cell by transfecting a plasmid. The hairpin cleaved off by enzyme to leave double stranded SiRNA.
Many online programmes are available to help provide the most suitable siRNA sequence from the mRNA sequence of the protein you wish to knock down.
These computer programmes 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. Reduces the cost of synthesis and siRNA duplex more resistant to exonuclease activity.
- GC Content
Ideally GC content < 50% (most software defaults range 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.
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 – duel staining so can also check effect on expression and cellular location of other proteins.
- 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 RTPCR?
- Have you used the correct endogenous positive and negative controls? Have you used scrambled SiRNA or standard negative siRNA controls?