Small interfering RNA and gene expression analysis using a multiplex branched DNA assay without RNA purification.
The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.
Zhang
Aiguo
A
Genospectra, Inc., Fremont 94555, CA. azhang@genospectra.com
Pastor
Larry
L
Nguyen
Quan
Q
Luo
Yuling
Y
Yang
Wen
W
Flagella
Michael
M
Chavli
Rajesh
R
Bui
Son
S
Nguyen
Cung Tuong
CT
Zheng
Zhi
Z
He
Weihai
W
McMaster
Gary
G
Witney
Frank
F
D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells
D016207:Cytokines; D017931:DNA Primers; D016209:Interleukin-8; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012313:RNA; D004247:DNA; D013755:Tetradecanoylphorbol Acetate