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

Rautio J, Barken KB, Lahdenperä J, Breitenstein A, Molin S, Neubauer P - Microb. Cell Fact. (2003)

Bottom Line: 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.Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioCentrum-DTU, Building 301, The Technical University of Denmark, DK-2800 Lyngby, Denmark. peter.neubauer@oulu.fi

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 x 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.

No MeSH data available.


Related in: MedlinePlus

Effect of cell lysate and total RNA on the signal of the solution hybridization. 1.5 × 1011 SUC2 in vitro transcribed RNA molecules were added to the hybridization solution containing crude cell lysate (○) or RNA extract (●) from 1 × 106 – 1 × 109 S. cerevisiae cells grown on glucose. The results are expressed as a percentage value of the signal compared to that obtained with the same amount of pure in vitro transcribed target SUC2 mRNA. Approximately 1.4 μg total RNA was extracted from 1 × 108 cells. The error bars show the ± SD of three parallel samples.
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Figure 4: Effect of cell lysate and total RNA on the signal of the solution hybridization. 1.5 × 1011 SUC2 in vitro transcribed RNA molecules were added to the hybridization solution containing crude cell lysate (○) or RNA extract (●) from 1 × 106 – 1 × 109 S. cerevisiae cells grown on glucose. The results are expressed as a percentage value of the signal compared to that obtained with the same amount of pure in vitro transcribed target SUC2 mRNA. Approximately 1.4 μg total RNA was extracted from 1 × 108 cells. The error bars show the ± SD of three parallel samples.

Mentions: The effect of biological material on the signal of the solution hybridization was studied by incubating various amounts (2 × 105 – 1 × 109) of lysed S. cerevisiae BY4737 cells or total RNA extract of the cells together with 1 × 1011 in vitro transcribed SUC2 mRNA target molecules. BY4743 yeast cells contained undetectable levels of SUC2 mRNA when grown on glucose (data not shown). No inhibition was observed when 106 or less lysed yeast cells or their RNA content were added to the hybridization (Fig. 4). 40 – 55% decrease in the signal of SUC2 mRNA was observed when 107 to 109 lysed cells were added to the hybridization solution. With extracted RNA the decrease was respectively 15 – 40%.


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

Rautio J, Barken KB, Lahdenperä J, Breitenstein A, Molin S, Neubauer P - Microb. Cell Fact. (2003)

Effect of cell lysate and total RNA on the signal of the solution hybridization. 1.5 × 1011 SUC2 in vitro transcribed RNA molecules were added to the hybridization solution containing crude cell lysate (○) or RNA extract (●) from 1 × 106 – 1 × 109 S. cerevisiae cells grown on glucose. The results are expressed as a percentage value of the signal compared to that obtained with the same amount of pure in vitro transcribed target SUC2 mRNA. Approximately 1.4 μg total RNA was extracted from 1 × 108 cells. The error bars show the ± SD of three parallel samples.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC156893&req=5

Figure 4: Effect of cell lysate and total RNA on the signal of the solution hybridization. 1.5 × 1011 SUC2 in vitro transcribed RNA molecules were added to the hybridization solution containing crude cell lysate (○) or RNA extract (●) from 1 × 106 – 1 × 109 S. cerevisiae cells grown on glucose. The results are expressed as a percentage value of the signal compared to that obtained with the same amount of pure in vitro transcribed target SUC2 mRNA. Approximately 1.4 μg total RNA was extracted from 1 × 108 cells. The error bars show the ± SD of three parallel samples.
Mentions: The effect of biological material on the signal of the solution hybridization was studied by incubating various amounts (2 × 105 – 1 × 109) of lysed S. cerevisiae BY4737 cells or total RNA extract of the cells together with 1 × 1011 in vitro transcribed SUC2 mRNA target molecules. BY4743 yeast cells contained undetectable levels of SUC2 mRNA when grown on glucose (data not shown). No inhibition was observed when 106 or less lysed yeast cells or their RNA content were added to the hybridization (Fig. 4). 40 – 55% decrease in the signal of SUC2 mRNA was observed when 107 to 109 lysed cells were added to the hybridization solution. With extracted RNA the decrease was respectively 15 – 40%.

Bottom Line: 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.Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioCentrum-DTU, Building 301, The Technical University of Denmark, DK-2800 Lyngby, Denmark. peter.neubauer@oulu.fi

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 x 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.

No MeSH data available.


Related in: MedlinePlus