1. DSpace at MEDIU
  2. Harvested articles مقالات مستوردة من مؤسسات وجامعات عالمية
  3. Digital Csic
Please use this identifier to cite or link to this item: http://dspace.mediu.edu.my:8181/xmlui/handle/10261/2882
Full metadata record
DC FieldValueLanguage
dc.creatorMüllner, Ernst W.-
dc.creatorPrete, M. Julieta Del-
dc.creatorVernal, Rolando-
dc.creatorDolznig, Helmut-
dc.creatorGarcía-Sanz, José A.-
dc.date2008-02-06T16:07:24Z-
dc.date2008-02-06T16:07:24Z-
dc.date2007-03-
dc.date.accessioned2017-01-31T00:59:59Z-
dc.date.available2017-01-31T00:59:59Z-
dc.identifierPMCID: 1800518-
dc.identifierRNA. 2007 March; 13(3): 414–421-
dc.identifierhttp://hdl.handle.net/10261/2882-
dc.identifier10.1261/rna.79407-
dc.identifier.urihttp://dspace.mediu.edu.my:8181/xmlui/handle/10261/2882-
dc.descriptionWe thank Drs. Carmen Rodriguez and Charles Theillet (INSERM, Montpellier) for providing tissue samples and Dr. Angel Zaballos (CNB, Madrid) for his help with the quantitative PCR reaction.-
dc.descriptionUsing cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA+ RNA. Aiming to use small quantities of solid tissue samples (10–20 mg/sample), polyA+ RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen–frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.-
dc.descriptionThe work in the authors’ laboratories was supported by grants from the U.S. Army Medical Research and Acquisition Activity (Breast Cancer Program) Contract number DAMD17-02-1-0339 (to J.A.G.-S.) and from the Spanish Ministry of Education and Science, contract SAF2003-00519 (to J.A.G.-S.), and the Austrian “Fonds zur Förderung der wissenschaftlichen Forschung, FWF” (to E.W.M.). R.V. is on leave from the Department of Conservative Odontology, Dentistry School, University of Chile. We also acknowledge the Unidad de Genomica, Parque Científico de Madrid, and Universidad Complutense de Madrid.-
dc.descriptionPeer reviewed-
dc.format461278 bytes-
dc.formatapplication/pdf-
dc.languageeng-
dc.publisherCambridge University Press-
dc.rightsopenAccess-
dc.subjectPolysome fractionation-
dc.subjectTissue samples-
dc.subjectSucrose gradients-
dc.subjectRT-PCR-
dc.subjectExpression profiling-
dc.titleIsolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments-
dc.typeArtículo-
Appears in Collections:Digital Csic

Files in This Item:
There are no files associated with this item.
Show simple item record


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.