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Plos One : C911 ; a Bench-level Control for Sequence Specific Sirna Off-target Effects, Volume 7

By Semsey, Szabolcs

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Book Id: WPLBN0003934614
Format Type: PDF eBook :
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Reproduction Date: 2015

Title: Plos One : C911 ; a Bench-level Control for Sequence Specific Sirna Off-target Effects, Volume 7  
Author: Semsey, Szabolcs
Volume: Volume 7
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection (Contemporary)
Historic
Publication Date:
Publisher: Plos

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Semsey, S. (n.d.). Plos One : C911 ; a Bench-level Control for Sequence Specific Sirna Off-target Effects, Volume 7. Retrieved from http://netlibrary.net/


Description
Description : Small interfering RNAs (siRNAs) have become a ubiquitous experimental tool for down-regulating mRNAs. Unfortunately, off-target effects are a significant source of false positives in siRNA experiments and an effective control for them has not previously been identified. We introduce two methods of mismatched siRNA design for negative controls based on changing bases in the middle of the siRNA to their complement bases. To test these controls, a test set of 20 highly active siRNAs (10 true positives and 10 false positives) was identified from a genome-wide screen performed in a cell-line expressing a simple, constitutively expressed luciferase reporter. Three controls were then synthesized for each of these 20 siRNAs, the first two using the proposed mismatch design methods and the third being a simple random permutation of the sequence (scrambled siRNA). When tested in the original assay, the scrambled siRNAs showed significantly reduced activity in comparison to the original siRNAs, regardless of whether they had been identified as true or false positives, indicating that they have little utility as experimental controls. In contrast, one of the proposed mismatch design methods, dubbed C911 because bases 9 through 11 of the siRNA are replaced with their complement, was able to completely distinguish between the two groups. False positives due to off-target effects maintained most of their activity when the C911 mismatch control was tested, whereas true positives whose phenotype was due to on-target effects lost most or all of their activity when the C911 mismatch was tested. The ability of control siRNAs to distinguish between true and false positives, if widely adopted, could reduce erroneous results being reported in the literature and save research dollars spent on expensive follow-up experiments.

 

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