This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thieme, D. Articles by Grass, G. Search for Related Content PubMed PubMed Citation Articles by Thieme, D. Articles by Grass, G. Agricola Articles by Thieme, D. Articles by Grass, G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2008, p. 7463-7470, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01370-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

Daniel Thieme,^1,2 Peter Neubauer,² Dietrich H. Nies,¹ and Gregor Grass^3*

Institut für Biologie/Mikrobiologie, Martin-Luther-Universität, Halle-Wittenberg, Germany,¹ Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter Oulu, University of Oulu, P.O. Box 4300, FI-90014 Oulu, Finland,² School of Biological Sciences, University of Nebraska, Lincoln, Nebraska³

Received 18 June 2008/ Accepted 18 October 2008

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.

---

* Corresponding author. Mailing address: School of Biological Sciences, Beadle Center, University of Nebraska, Lincoln, 1901 Vine St., Lincoln, NE 68588. Phone: (402) 472-2849. Fax: (402) 472-8722. E-mail: ggrass2{at}unlnotes.unl.edu

Published ahead of print on 24 October 2008.

---

Applied and Environmental Microbiology, December 2008, p. 7463-7470, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01370-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.