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Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion
M. Stastna, A. Thomas, J. Germano, S. Pourpirali, JE. Van Eyk, RA. Gottlieb,
Jazyk angličtina Země Spojené státy americké
Typ dokumentu časopisecké články, Research Support, N.I.H., Extramural, práce podpořená grantem, audiovizuální média
NLK
Free Medical Journals
od 2006 do 2018
PubMed Central
od 2006 do 2018
Europe PubMed Central
od 2006 do 2018
PubMed
30222143
DOI
10.3791/58079
Knihovny.cz E-zdroje
- MeSH
- messenger RNA genetika MeSH
- mikro RNA metabolismus MeSH
- myši MeSH
- polyribozomy metabolismus MeSH
- proteomika metody MeSH
- srdce růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.
Advanced Clinical Biosystems Research Institute Cedars Sinai Medical Center
Institute of Analytical Chemistry of the Czech Academy of Sciences
Citace poskytuje Crossref.org
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