RNA sequence analysis
Go to parent Basic bioinformatics concepts, databases and tools#Exercises_during_the_training
Analyzing a mRNA sequence for selecting siRNA to silence a gene
We will try to knock down the expression of a gene responsible for the aggregation of muscle cells by designing a siRNA to that gene.
The sarcolemmal membrane-associated protein (SLMAP gene) of mouse is known to be expressed in myoblasts and is believed to play a role in the fusion of myoblasts into myotubes. Suppose you want to test experimentally the role of this gene by performing an in vivo silencing experiment using a plasmid with an siRNA gene. We will use the tool of AMBION, which implements the Tuschl criteria to select a suitable target in the SLMAP mRNA.
| Find a suitable siRNA do silence the SLMAP gene in mouse for in vivo experiments. |
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There are three siRNA available for this mouse gene. You see that you can directly order the AMBION kits for these siRNAs.
| Check the position of the siRNA in the transcript. |
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You can click View details to get info on the position of the siRNA in the mRNA sequences for this gene.
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If you don't want to order a kit from AMBION for your silencing experiment, you might consider another tool since the output of AMBION is not very informative: you don't get easy access to the sequence of the miRNA for instance.
Follow this link to another siRNA tool that compared different sets of guidelines for selecting siRNAs using luciferase assays. Click here for an overview of the sets of guidelines that were compared and the results of the comparison.
| Retrieve the sequence of the SLMAP gene in mouse (accession: NM_032008). |
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Enter the RefSeq accession number in the appropriate box and click retrieve Sequence. This should automatically retrieve the RefSeq mRNA sequence of the slmap gene. |
Click the Click here button in the Options section to take a look at the parameters. The Minimization of seed-dependent off-target effects section allows you reduce the number of genes other than slmap whose expression will be downregulated by the siRNA because of unspecific binding. This unspecific binding is correlated with the stability of the duplex formed by the siRNA and the mRNA.</li>
| Define the database to check the specificity of the siRNAs, only show specific siRNAs and show how much aspecific targets are expected. |
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The Specificity check section allows you to check upfront if the suggested siRNA is binding specifically to the slmap mRNA (similar to primerBLAST)
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Select to Only show siRNAs that match all checked criteria and click Design siRNA.
There are many siRNAs for this mouse gene. You see a lot of info: location, sequence and specificity of the selected siRNAs.
Warning: The presence of the motif 5'-UGGC-3' in the siRNA duplex was found to lead to strong cell toxicity (Fedorov et al., 2006)
