RT Journal Article
SR Electronic
T1 Luciferase shRNA Presents off-Target Effects on Voltage-Gated Ion Channels in Mouse Hippocampal Pyramidal Neurons
JF eneuro
JO eNeuro
FD Society for Neuroscience
SP ENEURO.0186-17.2017
DO 10.1523/ENEURO.0186-17.2017
VO 4
IS 5
A1 Yuto Hasegawa
A1 Wenjie Mao
A1 Sucharita Saha
A1 Georgia Gunner
A1 Jenya Kolpakova
A1 Gilles E. Martin
A1 Kensuke Futai
YR 2017
UL http://www.eneuro.org/content/4/5/ENEURO.0186-17.2017.abstract
AB 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.