Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

Jari Rautio, K. B. Barken, Juhani Lahdenperä, A. Breitenstein, S. Molin, P. Neubauer (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

43 Citations (Scopus)

Abstract

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.
Original languageEnglish
Number of pages9
JournalMicrobial Cell Factories
Volume2
Issue number4
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

RNA
Yeast
Assays
Yeasts
Messenger RNA
Molecules
Saccharomyces cerevisiae
beta-Fructofuranosidase
Ribosomal RNA
Streptavidin
Phosphatases
Enzyme activity
Cell growth
Growth
Biotin
Alkaline Phosphatase
Fluorescence
Monitoring
Enzymes

Cite this

Rautio, Jari ; Barken, K. B. ; Lahdenperä, Juhani ; Breitenstein, A. ; Molin, S. ; Neubauer, P. / Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. In: Microbial Cell Factories. 2003 ; Vol. 2, No. 4.
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abstract = "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.",
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Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. / Rautio, Jari; Barken, K. B.; Lahdenperä, Juhani; Breitenstein, A.; Molin, S.; Neubauer, P. (Corresponding Author).

In: Microbial Cell Factories, Vol. 2, No. 4, 2003.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

AU - Rautio, Jari

AU - Barken, K. B.

AU - Lahdenperä, Juhani

AU - Breitenstein, A.

AU - Molin, S.

AU - Neubauer, P.

PY - 2003

Y1 - 2003

N2 - 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.

AB - 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.

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VL - 2

JO - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

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