Document Detail


A multiplex branched DNA assay for parallel quantitative gene expression profiling.
MedLine Citation:
PMID:  16545767     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.
Authors:
Michael Flagella; Son Bui; Zhi Zheng; Cung Tuong Nguyen; Aiguo Zhang; Larry Pastor; Yunqing Ma; Wen Yang; Kimberly L Crawford; Gary K McMaster; Frank Witney; Yuling Luo
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Publication Detail:
Type:  Journal Article     Date:  2006-03-02
Journal Detail:
Title:  Analytical biochemistry     Volume:  352     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2006 May 
Date Detail:
Created Date:  2006-04-24     Completed Date:  2006-08-08     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  50-60     Citation Subset:  IM    
Affiliation:
Genospectra, Fremont, CA 94555, USA.
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MeSH Terms
Descriptor/Qualifier:
Alternative Splicing
Apoptosis
Branched DNA Signal Amplification Assay / methods*
Fluorescent Dyes
Gene Expression Profiling / methods*
Gene Expression Regulation
Hela Cells
Humans
Inflammation / metabolism
Models, Biological
Nucleic Acid Hybridization
Oligonucleotide Probes
RNA, Messenger / analysis,  metabolism
U937 Cells
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/Oligonucleotide Probes; 0/RNA, Messenger

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


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