CDS vs. 3′ UTR In some cases, it may be preferable to only target the 3′ UTR of an mRNA. For example, the CDS may be highly similar to a related paralog gene that should not be co-targeted. Or a rescue experiment will be performed, and one would rather use the native CDS for rescue. (Note that we can also provide rescue constructs for siPOOLs that target CDS. The rescue sequence uses the most common alternative codon in the targeted Read More
One advantage of pooling siRNAs is that the pool tends to silence about as well as any single siRNA. That is why independent complex siRNA pools (siPOOLs) for the same gene have much more similar and better average silencing than single siRNAs or mini-pools (Dharmacon), as discussed in our last blog post. Another advantage of complex siRNA pools is that you can cover more regions of the target gene. This may be especially important when silencing lncRNAs, which can be Read More
Although we talk a lot about off-targets, one of the main advantages of siPOOLs (complex siRNA pools) compared to single siRNAs or mini-pools (Dharmacon) is that they provide near optimum silencing of target genes. Two siPOOLs for the same gene give very similar knock down levels, and their silencing is around the best of any single siRNA. Given how many candidate siRNAs there are for a gene, and how difficult it is to accurately predict silencing levels, this makes siPOOLs Read More
The C911 siRNA control generated a lot of excitement in the RNAi world when it emerged ~11 years ago. A former colleague, who was a pioneer in the commercialisation of RNAi, described it then as the biggest breakthrough in the last 10 years of RNAi research. The idea of the C911 control is to get rid of the on-target effect of the siRNA by using the complement of bases 9-11, while retaining any off-target (seed-based) effects of the siRNA, which Read More
At siTOOLs we are celebrating RNA day (Aug. 1st) the entire month with a focus on all things RNA and a promotion on siPOOLs and riboPOOLs. RNA day is celebrated the 1st of August, since the RNA codon that initiates protein synthesis is made of the following nucleotides: adenine (A), uracil (U), and guanine (G). AUG codes for the amino acid methionine (Met) in eukaryotes and formyl methionine (fMet) in prokaryotes. RNA is one of the most versatile biomolecules in Read More
Which technology is better, RNAi or CRISPR? The best answer to this question, like so many others is, it depends. If cells can adapt and compensate for loss of the gene, or you want to titrate gene levels (important in drug discovery), then RNAi will be better. If a gene’s transcripts have lots of secondary structure and must be silenced to 99.9% in order to see an assay phenotype, then CRISPR may be better. We have used two large datasets Read More
In the last post, we saw that chemically modified ON-TARGETplus siRNAs still have a strong seed effect. The seed-based off-target effects (measured by correlation of reagents with the same 7mer seed) were as strong for chemically modified ON-TARGETplus (R = 0.50) and Silencer Select (R = 0.59) as what we typically see with unmodified siRNAs (Qiagen, siGENOME, or Silencer). Chemical modification must not prevent seed-based target recognition, because RISC uses the seed to scan the transcriptome for target sites. Because Read More
History of chemical modifications Chemical modification has long been proposed as a way to limit the off-target effects of siRNAs. The earliest siRNAs from the two main commercial suppliers (siGENOME from Dharmacon/Horizon Discovery, and Silencer from Ambion/ThermoFisher) were quickly replaced with new chemically-modified siRNAs (ON-TARGETplus from Dharmacon, and Silencer Select from Ambion). We have already seen that Silencer Select siRNAs, despite their chemical modification, maintain a strong off-target seed effect. The phenotypic correlation between siGENOME (unmodified) and ON-TARGETplus (chemically modified) Read More
Bacteroides thetaiotaomicron is a commensal bacterium that inhabits primarily the human large intestine and is considered one of the most important members of this microbial community. B. thetaiotaomicron is a highly versatile microbe, capable of utilizing a wide range of carbohydrates including those that are indigestible by human enzymes. It breaks down complex polysaccharides from plant cell walls and other dietary sources, producing short-chain fatty acids (SCFAs) that are an important energy source for humans. Furthermore, it has also been Read More
Microbiomes are the diverse communities of microorganisms that inhabit different parts of our bodies, as well as the environment around us. In recent years, research has revealed the vast and complex hidden world of microbiomes and their impact on our lives, from influencing our digestion and immune system to potentially affecting our mood and behavior. Advances in technology have enabled scientists to study microbiomes in unprecedented detail, leading to new insights into their diversity and functions. Understanding the microbiome and Read More
