University of Massachusetts Medical School Faculty Publications
UMMS Affiliation
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry; Martin Lab; Futai Lab; Graduate School of Biomedical Sciences
Publication Date
2017-10-11
Document Type
Article
Disciplines
Neuroscience and Neurobiology
Abstract
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.
Keywords
ion channels, luciferase, off-target effects, RNA interference, short hairpin
Rights and Permissions
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
10.1523/ENEURO.0186-17.2017
Source
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
Related Resources
Journal/Book/Conference Title
eNeuro
PubMed ID
29034317
Repository Citation
Hasegawa Y, Mao W, Saha S, Gunner G, Kolpakova J, Martin GE, Futai K. (2017). Luciferase shRNA Presents off-Target Effects on Voltage-Gated Ion Channels in Mouse Hippocampal Pyramidal Neurons. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1523/ENEURO.0186-17.2017. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1453
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.