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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

Level of 18S rRNA of S. cerevisiae carlsbergensis determined by sandwich hybridization as molecules per cell (●) in different growth states. The growth was followed by measuring the optical density at 600 nm (○) and the specific growth rate μ (□) was determined. The yeast was cultured in medium containing 1% yeast extract, 2% Bacto peptone solution and 2% glucose as carbon source. 1 × 106 – 3 × 106 yeast cells as crude lysate was added to sandwich hybridization for 18S rRNA analysis. OD600 = 1 corresponds to 5.5 × 108yeast cells ml-1. The error bars of show the ± SD of three parallel samples.
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Figure 5: Level of 18S rRNA of S. cerevisiae carlsbergensis determined by sandwich hybridization as molecules per cell (●) in different growth states. The growth was followed by measuring the optical density at 600 nm (○) and the specific growth rate μ (□) was determined. The yeast was cultured in medium containing 1% yeast extract, 2% Bacto peptone solution and 2% glucose as carbon source. 1 × 106 – 3 × 106 yeast cells as crude lysate was added to sandwich hybridization for 18S rRNA analysis. OD600 = 1 corresponds to 5.5 × 108yeast cells ml-1. The error bars of show the ± SD of three parallel samples.

Mentions: The inhibition effect of cell lysate on the hybridization was taken in to consideration, when the signal of target RNA in sandwich hybridization was quantified in growth experiments (see Fig. 5 and 6).


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)

Level of 18S rRNA of S. cerevisiae carlsbergensis determined by sandwich hybridization as molecules per cell (●) in different growth states. The growth was followed by measuring the optical density at 600 nm (○) and the specific growth rate μ (□) was determined. The yeast was cultured in medium containing 1% yeast extract, 2% Bacto peptone solution and 2% glucose as carbon source. 1 × 106 – 3 × 106 yeast cells as crude lysate was added to sandwich hybridization for 18S rRNA analysis. OD600 = 1 corresponds to 5.5 × 108yeast cells ml-1. The error bars of show the ± SD of three parallel samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC156893&req=5

Figure 5: Level of 18S rRNA of S. cerevisiae carlsbergensis determined by sandwich hybridization as molecules per cell (●) in different growth states. The growth was followed by measuring the optical density at 600 nm (○) and the specific growth rate μ (□) was determined. The yeast was cultured in medium containing 1% yeast extract, 2% Bacto peptone solution and 2% glucose as carbon source. 1 × 106 – 3 × 106 yeast cells as crude lysate was added to sandwich hybridization for 18S rRNA analysis. OD600 = 1 corresponds to 5.5 × 108yeast cells ml-1. The error bars of show the ± SD of three parallel samples.
Mentions: The inhibition effect of cell lysate on the hybridization was taken in to consideration, when the signal of target RNA in sandwich hybridization was quantified in growth experiments (see Fig. 5 and 6).

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