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Applications of RNA interference high-throughput screening technology in cancer biology and virology.

Gao S, Yang C, Jiang S, Xu XN, Lu X, He YW, Cheung A, Wang H - Protein Cell (2014)

Bottom Line: RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function.This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight.Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, John Radcliffe Hospital, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK, shan.gao@oncology.ox.ac.uk.

ABSTRACT
RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi high-throughput screening (HTS) has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

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Related in: MedlinePlus

RNAi high-throughput screening approaches. (A) Arrayed screen using siRNA in a multiwell plate. siRNA can be reverse-transfected into cells for 48–72 h. Then, cells can be selected using some pressure or left for a longer time period to develop phenotypes, which can be determined using various readouts. (B) Pooled screen using pooled shRNA viral particles to infect target cells. After 48 h, cells can be divided into multiple groups. One group can be frozen as an initial population. Other groups can be treated with various pressures for some period. Then, relative enrichment of shRNAs can be analyzed using next generation sequencing
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Fig2: RNAi high-throughput screening approaches. (A) Arrayed screen using siRNA in a multiwell plate. siRNA can be reverse-transfected into cells for 48–72 h. Then, cells can be selected using some pressure or left for a longer time period to develop phenotypes, which can be determined using various readouts. (B) Pooled screen using pooled shRNA viral particles to infect target cells. After 48 h, cells can be divided into multiple groups. One group can be frozen as an initial population. Other groups can be treated with various pressures for some period. Then, relative enrichment of shRNAs can be analyzed using next generation sequencing

Mentions: In the individual siRNA screening format, in theory, different siRNAs targeting the same mRNA species should induce an identical phenotype. In reality, these siRNAs rarely lead to the same phenotype because different siRNAs are heterogeneous in inducing Dicer-mediated degradation of the target mRNA. Furthermore, a number of phenotypes only occur while the target is depleted to a certain level. Therefore, if any two individual siRNAs can confirm the same phenotype, they are considered as the final hits. Occasionally, a high proportion of a single siRNA hit may be ignored and subsequently lead to false negatives in the same assay (Fig. 2A) (Iorns et al., 2007).Figure 2


Applications of RNA interference high-throughput screening technology in cancer biology and virology.

Gao S, Yang C, Jiang S, Xu XN, Lu X, He YW, Cheung A, Wang H - Protein Cell (2014)

RNAi high-throughput screening approaches. (A) Arrayed screen using siRNA in a multiwell plate. siRNA can be reverse-transfected into cells for 48–72 h. Then, cells can be selected using some pressure or left for a longer time period to develop phenotypes, which can be determined using various readouts. (B) Pooled screen using pooled shRNA viral particles to infect target cells. After 48 h, cells can be divided into multiple groups. One group can be frozen as an initial population. Other groups can be treated with various pressures for some period. Then, relative enrichment of shRNAs can be analyzed using next generation sequencing
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4225462&req=5

Fig2: RNAi high-throughput screening approaches. (A) Arrayed screen using siRNA in a multiwell plate. siRNA can be reverse-transfected into cells for 48–72 h. Then, cells can be selected using some pressure or left for a longer time period to develop phenotypes, which can be determined using various readouts. (B) Pooled screen using pooled shRNA viral particles to infect target cells. After 48 h, cells can be divided into multiple groups. One group can be frozen as an initial population. Other groups can be treated with various pressures for some period. Then, relative enrichment of shRNAs can be analyzed using next generation sequencing
Mentions: In the individual siRNA screening format, in theory, different siRNAs targeting the same mRNA species should induce an identical phenotype. In reality, these siRNAs rarely lead to the same phenotype because different siRNAs are heterogeneous in inducing Dicer-mediated degradation of the target mRNA. Furthermore, a number of phenotypes only occur while the target is depleted to a certain level. Therefore, if any two individual siRNAs can confirm the same phenotype, they are considered as the final hits. Occasionally, a high proportion of a single siRNA hit may be ignored and subsequently lead to false negatives in the same assay (Fig. 2A) (Iorns et al., 2007).Figure 2

Bottom Line: RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function.This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight.Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, John Radcliffe Hospital, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK, shan.gao@oncology.ox.ac.uk.

ABSTRACT
RNA interference (RNAi) is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi high-throughput screening (HTS) has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

Show MeSH
Related in: MedlinePlus