Protocol for in vitro transcribing mRNAs with defined poly(A)-tail lengths and visualizing sequential PABP binding.
| Autor: | Grandi C; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands., Emmaneel M; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands., Nelissen FHT; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands., Hansen MMK; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands. Electronic address: maike.hansen@ru.nl. |
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| Jazyk: | angličtina |
| Zdroj: | STAR protocols [STAR Protoc] 2024 Aug 30; Vol. 5 (3), pp. 103284. Date of Electronic Publication: 2024 Aug 30. |
| DOI: | 10.1016/j.xpro.2024.103284 |
| Abstrakt: | Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure. For complete details on the use and execution of this protocol, please refer to Grandi et al. 1 . Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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