3 replies to this topic

[claritylight]

I can't find a good definition or description online about what it means when a study does a depletion or enrichment screening of shRNAs. Do they  enrich or deplete the shRNAs or the genes? I assume its the shRNA for the sake of this post. What I understand so far is if a study does either of these screening, the shRNAs get enriched or depleted and the investigator looks at the cell phenotype to see how viable the gene target is to the cell, so to me it sounds similar to how one would use a drug and do a dosage study (different dosages of a drug treatment) to see how it affects cell viability and phenotype. If the shRNAs are getting depleted in a study, does that mean the gene won't get knocked down and if the shRNAs are enriched, then the gene will certainly be knocked down?

I've also been reading about positive selection versus negative selection shRNA screening. Are these names the same as saying the shRNA has gone through enrichment or depletion screening ?

Can someone give me information on the web to read involving shRNA enrichment and depletion studies, I just can't find any it seems!

I assume the above questions can also be applicable to other RNAi applications like siRNAs and miRNAs as well?

[pcrman]

Hi claritylight,

shRNA library screening works this way: a library of shRNAs that targets all genes or a group of genes are used to infect cells at a certain MOI so that one cell will only be infected by one viral particle. If the researcher is interested in identifying certain genes whose knockdown will cause a particular phenotype, eg, M phase arrest. After the shRNA infection, the researcher would enrich M phase cells by FACS analysis and recover the shRNA from the enriched cells. By identifying which shRNA(s) are enriched in the M phase cells, the target gene (one or more) that promote M phase is then  identified. This make perfect sense if the knockdown causes phenotypes which could be used to enrich the infected cells. If the knockdown causes depletion of infected cells, recovering the responsible shRNA is very complicated involving using many control treatments.


This page gives pretty good definitions:

Quote

A positive selection screen allows identification of an shRNA whose expression allows a cell to be isolated from a complex population of cells e.g. survival or selectable phenotype. Negative selection (dropout) screening identifies shRNA whose expression modulates the growth of cells and captures both positive and negative effects on the growth of a complex population of cells.  

[claritylight]

thanks so much! this really does help!

[claritylight]

pcrman, another question: what does it mean when a study has done genome-wide shRNA/siRNA screening? does this simply mean they have multiple shRNAs/siRNAs targeting multiple genes or gene family in the genome? can you guide me to a website that explains it? I assume this is just to provide higher throughput of shRNA/siRNA screening, instead of just doing one gene knockdown at a time but I don't know if there is more detail than this.