High-throughput function-based gene identification using enzymatically generated short hairpin RNA library and massive parallel sequencing

Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.

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Faculty Opinions recommendation of Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010575.372761 ◽

2006 ◽

Author(s):

Santa Jeremy Ono

Keyword(s):

Mast Cells ◽

Cell Survival ◽

Rna Silencing ◽

Short Hairpin Rna ◽

Cutting Edge ◽

Cytokine Secretion ◽

Hairpin Rna ◽

Human Mast Cells ◽

Short Hairpin

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Faculty Opinions recommendation of Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032558.489061 ◽

2006 ◽

Author(s):

Vitaly Citovsky

Keyword(s):

Rna Interference ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Short Hairpin

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Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284342 ◽

2006 ◽

Vol 11 (3) ◽

pp. 236-246 ◽

Cited By ~ 6

Author(s):

Laurence H. Lamarcq ◽

Bradley J. Scherer ◽

Michael L. Phelan ◽

Nikolai N. Kalnine ◽

Yen H. Nguyen ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Strong Support ◽

Gc Content ◽

Rapid Identification ◽

Hairpin Rna ◽

Rna Sequences ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

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