Document Detail


Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis.
MedLine Citation:
PMID:  13679029     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-->3' direction; the appropriate template for any gene expression analysis.
Authors:
Mangalathu S Rajeevan; Irina M Dimulescu; Suzanne D Vernon; Mukesh Verma; Elizabeth R Unger
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Genomics     Volume:  82     ISSN:  0888-7543     ISO Abbreviation:  Genomics     Publication Date:  2003 Oct 
Date Detail:
Created Date:  2003-09-18     Completed Date:  2004-06-01     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8800135     Medline TA:  Genomics     Country:  United States    
Other Details:
Languages:  eng     Pagination:  491-7     Citation Subset:  IM    
Affiliation:
Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, US Department of Health and Human Services, Atlanta, GA 30333, USA. mor4@cdc.gov
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MeSH Terms
Descriptor/Qualifier:
Cell Line
DNA, Complementary / biosynthesis
Gene Amplification
Gene Expression
Gene Expression Profiling / methods*
Humans
RNA / biosynthesis*
RNA, Messenger*
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Grant Support
ID/Acronym/Agency:
Y1-CN-0101-01/CN/NCI NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Complementary; 0/RNA, Messenger; 63231-63-0/RNA; EC 2.7.7.49/RNA-Directed DNA Polymerase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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