University of Massachusetts Medical School Faculty Publications

UMMS Affiliation

Brudnick Neuropsychiatric Research Institute, Department of Psychiatry; Martin Lab; Futai Lab

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Document Type



Neuroscience and Neurobiology


RNA interference (RNAi) is a straightforward approach to study gene function from the in vitro cellular level to in vivo animal behavior. Although RNAi-mediated gene knockdown has become essentially routine in neuroscience over the past ten years, off-target effects of short hairpin RNAs (shRNAs) should be considered as the proper choice of control shRNA is critical in order to perform meaningful experiments. Luciferase shRNA (shLuc), targeting firefly luciferase, and scrambled shRNAs (shScrs) have been widely used as controls for vertebrate cell research. However, thorough validation of control shRNAs has not been made to date. Here, we performed thorough physiological and morphological studies against control shRNAs in mouse hippocampal CA1 pyramidal neurons. As expected, all control shRNAs exhibited normal basal synaptic transmission and dendritic morphology. However, to our surprise, shLuc exerted severe off-target effects on voltage-gated ion channel function, while the shScr had no detectable changes. These results indicate that thorough validation of shRNA is imperative and, in the absence of such validation, that shScr is the best available negative control for gene knockdown studies.


ion channels, luciferase, off-target effects, RNA interference, short hairpin

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Copyright © 2017 Hasegawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

DOI of Published Version



eNeuro. 2017 Oct 11;4(5). pii: ENEURO.0186-17.2017. doi: 10.1523/ENEURO.0186-17.2017. eCollection 2017 Sep-Oct. Link to article on publisher's site

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Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.



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