This Article Full Text (PDF) All Versions of this Article: 1087057105277414v1 10/6/549    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Zhang, A. Articles by Witney, F. Search for Related Content PubMed PubMed Citation Articles by Zhang, A. Articles by Witney, F. Social Bookmarking                   What's this?

Small Interfering RNA and Gene Expression Analysis Using a Multiplex Branched DNA Assay without RNA Purification

Genospectra, Inc., Fremont, CA

Larry Pastor

Genospectra, Inc., Fremont, CA

Quan Nguyen

Genospectra, Inc., Fremont, CA

Yuling Luo

Genospectra, Inc., Fremont, CA

Wen Yang

Genospectra, Inc., Fremont, CA

Michael Flagella

Genospectra, Inc., Fremont, CA

Rajesh Chavli

Genospectra, Inc., Fremont, CA

Son Bui

Genospectra, Inc., Fremont, CA

Cung Tuong Nguyen

Genospectra, Inc., Fremont, CA

Zhi Zheng

Genospectra, Inc., Fremont, CA

Weihai He

Genospectra, Inc., Fremont, CA

Gary McMaster

Genospectra, Inc., Fremont, CA

Frank Witney

Genospectra, Inc., Fremont, CA

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-1 (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.

Key Words: multiplex branched DNA • gene expression • siRNA • cytokines • Luminex

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Z. Zheng, Y. Luo, and G. K. McMaster Sensitive and Quantitative Measurement of Gene Expression Directly from a Small Amount of Whole Blood Clin. Chem., July 1, 2006; 52(7): 1294 - 1302. [Abstract] [Full Text] [PDF]