Microbial Cell Factories

Background: A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules. Results: The sensitivity of the assay was approximately 1.2 × 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities. Conclusions: The here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions. Background Development of novel methods for fast, sensitive and reliable RNA quantification has recently got increasing attention. Conventional methods for RNA analysis, like Northern and slot blot hybridization are in general time consuming, laborious and allow only relative quantification within a narrow concentration range. More novel methods for RNA analysis e.g. RT-PCR and real time RTPCR are highly sensitive, but also slightly susceptible to experimental interferences, like template inhibition due to insufficient purification [2] and lack accuracy for quantification due to biases connected to PCR and reverse transcription reactions, which in general are accepted errors connected to these methods. The sandwich hybridization method is a suitable alternative for RNA quantification. The method is based on the detection of hybridization events between two specific Published: 28 April 2003 Microbial Cell Factories 2003, 2:4 Received: 20 March 2003 Accepted: 28 April 2003 This article is available from: http://www.microbialcellfactories.com/content/2/1/4 © 2003 Rautio et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/4