Julie Ry Gustafsson1, Georgia Katsioudi2, Shohreh Issazadeh-Navikas3, Birgitte Rahbek Kornum4. 1. Department of Clinical Biochemistry, Molecular Sleep Laboratory, Rigshospitalet, Nordre Ringvej 57, 2600 Glostrup, Denmark. Electronic address: julie-jacobsen@hotmail.com. 2. Department of Clinical Biochemistry, Molecular Sleep Laboratory, Rigshospitalet, Nordre Ringvej 57, 2600 Glostrup, Denmark. Electronic address: gkatsioudi@gmail.com. 3. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark. Electronic address: shohreh.issazadeh@bric.ku.dk. 4. Department of Clinical Biochemistry, Molecular Sleep Laboratory, Rigshospitalet, Nordre Ringvej 57, 2600 Glostrup, Denmark; Department of Neurophysiology, Rigshospitalet, Glostrup, Denmark. Electronic address: birgitte.kornum@regionh.dk.
Abstract
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.
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.
Authors: Colleen M Bartman; Kimberly E Stelzig; David R Linden; Y S Prakash; Sergio E Chiarella Journal: Am J Physiol Lung Cell Mol Physiol Date: 2021-05-26 Impact factor: 6.011