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Studying BDNF/TrkB Signaling: High-Throughput Microfluidic Gene Expression Analysis from Rare or Limited Samples of Adult and Aged Central Neurons
High-throughput next generation sequencing technologies are an invaluable tool to gain insight into the transcriptional states of large cohorts of cells. Such data can help to shed light on the organization of tissues and pathways under normal and pathological conditions. In our case, we are using the above technology to decipher how the enkephalinergic medium spiny neurons (MSNs) of the striatum adapt to aging in the presence or absence of BDNF/TrkB signaling. However, sequencing data must be validated, ideally with an alternative method that interrogates the transcriptional state of cells, and is able to detect gene expression in rare single cells or bulk cells with high sensitivity. Thus, we have optimized a protocol for high-throughput microfluidic analysis [Fluidigm Dynamic Array Integrated Microfluidic Circuit (IFC)] to validate RNA sequencing data from a limited number of adult and aged sorted neurons. Here is a detailed description of this protocol.
Keywords:Bulk cell Fluidigm Gene expression Heatmap High-throughput Microfluidic Quantitative RNA sequencing RT-PCR TaqMan Validation
We would like to thank members of the Minichiello and the Nerlov laboratories for the support they provided. A.R.N. was supported by a Clarendon Scholarship. F.G. was supported by a China Scholarship Council (CSC). This work was supported by a BBSRC grant (BB/L021382/1) to L.M.
- 2.Besusso D, Geibel M, Kramer D, Schneider T, Pendolino V, Picconi B, Calabresi P, Bannerman DM, Minichiello L (2013) BDNF-TrkB signaling in striatopallidal neurons controls inhibition of locomotor behavior. Nat Commun 4:2031. https://doi.org/10.1038/ncomms3031 ADSCrossRefPubMedPubMedCentralGoogle Scholar
- 7.Drissen R, Buza-Vidas N, Woll P, Thongjuea S, Gambardella A, Giustacchini A, Mancini E, Zriwil A, Lutteropp M, Grover A, Mead A, Sitnicka E, Jacobsen SE, Nerlov C (2016) Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing. Nat Immunol 17:666–676. https://doi.org/10.1038/ni.3412 CrossRefPubMedPubMedCentralGoogle Scholar