A multiplex branched DNA assay for parallel quantitative gene expression profiling.
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.
Flagella
Michael
M
Genospectra, Fremont, CA 94555, USA.
Bui
Son
S
Zheng
Zhi
Z
Nguyen
Cung Tuong
CT
Zhang
Aiguo
A
Pastor
Larry
L
Ma
Yunqing
Y
Yang
Wen
W
Crawford
Kimberly L
KL
McMaster
Gary K
GK
Witney
Frank
F
Luo
Yuling
Y
D017398:Alternative Splicing; D017209:Apoptosis; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020298:U937 Cells
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger