Program in Molecular Medicine
Amino Acids, Peptides, and Proteins | Biochemistry, Biophysics, and Structural Biology | Cells | Congenital, Hereditary, and Neonatal Diseases and Abnormalities | Genetic Phenomena | Nervous System Diseases | Nucleic Acids, Nucleotides, and Nucleosides
Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for RNase digestion. We performed RNase titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling for low-input brain tissue.
Ribosomes, Protein Translation
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Copyright The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
DOI of Published Version
Nucleic Acids Res. 2019 Mar 18;47(5):e25. doi: 10.1093/nar/gky1292. Link to article on publisher's site
Nucleic acids research
Liu, Botao; Molinaro, Gemma; Shu, Huan; Stackpole, Emily E.; Huber, Kimberly M.; and Richter, Joel D., "Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice" (2019). Open Access Articles. 3807.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Amino Acids, Peptides, and Proteins Commons, Biochemistry, Biophysics, and Structural Biology Commons, Cells Commons, Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Genetic Phenomena Commons, Nervous System Diseases Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons