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Article: A critical assessment of the RNAse protection assay as a means of determining exon sizes

TitleA critical assessment of the RNAse protection assay as a means of determining exon sizes
Authors
Issue Date1993
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yabio
Citation
Analytical Biochemistry, 1993, v. 209 n. 2, p. 360-366 How to Cite?
AbstractThe RNAse protection assay is a highly sensitive assay which is commonly used to detect specific hybridization between complementary RNAs and to determine exon sizes in gene characterization studies. Unfortunately, each of the numerous steps involved in the assay could give artifacts depending on the probe used. In this study, common causes of artifacts have been identified using riboprobes which identify exons of known sizes. The RNAse concentration and duration of digestion used were found to be critical factors affecting exon size estimations. Five different riboprobes were tested to obtain a consensus optimum RNAse condition - 10 μg/ml RNAse A, 0.5 μg/ml RNAse T1 - enabling the correct determination of exon sizes. This condition was further analyzed for its specificity when RNAse protection assays were performed between highly homologous RNA fragments from two different species. Results show that this concentration of RNAse would efficiently cleave a minimum of two nucleotide mismatches. Single nucleotide mismatches were frequently not cleaved by the same RNAse concentration making it possible to detect the correct exon size regardless of such sequence polymorphisms in gene sequences.
Persistent Identifierhttp://hdl.handle.net/10722/147374
ISSN
2015 Impact Factor: 2.243
2015 SCImago Journal Rankings: 0.725
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorLau, ETen_US
dc.contributor.authorKong, RYCen_US
dc.contributor.authorCheah, KSEen_US
dc.date.accessioned2012-05-29T06:03:16Z-
dc.date.available2012-05-29T06:03:16Z-
dc.date.issued1993en_US
dc.identifier.citationAnalytical Biochemistry, 1993, v. 209 n. 2, p. 360-366en_US
dc.identifier.issn0003-2697en_US
dc.identifier.urihttp://hdl.handle.net/10722/147374-
dc.description.abstractThe RNAse protection assay is a highly sensitive assay which is commonly used to detect specific hybridization between complementary RNAs and to determine exon sizes in gene characterization studies. Unfortunately, each of the numerous steps involved in the assay could give artifacts depending on the probe used. In this study, common causes of artifacts have been identified using riboprobes which identify exons of known sizes. The RNAse concentration and duration of digestion used were found to be critical factors affecting exon size estimations. Five different riboprobes were tested to obtain a consensus optimum RNAse condition - 10 μg/ml RNAse A, 0.5 μg/ml RNAse T1 - enabling the correct determination of exon sizes. This condition was further analyzed for its specificity when RNAse protection assays were performed between highly homologous RNA fragments from two different species. Results show that this concentration of RNAse would efficiently cleave a minimum of two nucleotide mismatches. Single nucleotide mismatches were frequently not cleaved by the same RNAse concentration making it possible to detect the correct exon size regardless of such sequence polymorphisms in gene sequences.en_US
dc.languageengen_US
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yabioen_US
dc.relation.ispartofAnalytical Biochemistryen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshExonsen_US
dc.subject.meshHumansen_US
dc.subject.meshKineticsen_US
dc.subject.meshMiceen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshNeurofilament Proteins - Genetics - Isolation & Purificationen_US
dc.subject.meshRna Probesen_US
dc.subject.meshRna, Messenger - Analysisen_US
dc.subject.meshRibonucleases - Chemistryen_US
dc.subject.meshSensitivity And Specificityen_US
dc.titleA critical assessment of the RNAse protection assay as a means of determining exon sizesen_US
dc.typeArticleen_US
dc.identifier.emailCheah, KSE:hrmbdkc@hku.hken_US
dc.identifier.authorityCheah, KSE=rp00342en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1006/abio.1993.1135en_US
dc.identifier.pmid8470811-
dc.identifier.scopuseid_2-s2.0-0027157377en_US
dc.identifier.volume209en_US
dc.identifier.issue2en_US
dc.identifier.spage360en_US
dc.identifier.epage366en_US
dc.identifier.isiWOS:A1993KR44400027-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLau, ET=7103086081en_US
dc.identifier.scopusauthoridKong, RYC=7005290687en_US
dc.identifier.scopusauthoridCheah, KSE=35387746200en_US

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