Neurobasal media facilitates increased specificity of siRNA-mediated knockdown in primary cerebellar cultures.
BACKGROUND
Efficient and specific knockdown of proteins in post-mitotic cells such as differentiated neurons can be difficult to achieve. Further, special care must be taken to maintain the health of neurons in vitro. We wanted to achieve knockdown in primary cerebellar granule neurons, which can be effectively grown in Neurobasal™ media.


NEW METHOD
We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer.


RESULTS
We observed a more specific knockdown of target in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media.


COMPARISON WITH EXISTING METHODS
Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects.


CONCLUSIONS
Specific knockdown of target can be achieved in primary cerebellar granule cells using Accell siRNAs in Neurobasal™ media. This method ensures specific knockdown in post-mitotic neurons without the need for biosafety level 2 laboratories, additional reagents, or instruments needed by other transfection.
['Gustafsson|Julie Ry|JR|', 'Katsioudi|Georgia|G|', 'Issazadeh-Navikas|Shohreh|S|', 'Kornum|Birgitte Rahbek|BR|']
D000818:Animals D000831:Animals, Newborn D002470:Cell Survival D002478:Cells, Cultured D002531:Cerebellum D003470:Culture Media D021983:Cyclophilins D000076123:DNA (Cytosine-5-)-Methyltransferase 1 D004248:DNA (Cytosine-5-)-Methyltransferases D000090205:DNA Methyltransferase 3A D015536:Down-Regulation D005904:Glial Fibrillary Acidic Protein D051379:Mice D008807:Mice, Inbred BALB C D009447:Neuroblastoma D009474:Neurons D012333:RNA, Messenger D034741:RNA, Small Interfering D014404:Tubulin
Neurobasal media Neuron siRNA Transfection