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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

Comparison of extracted total RNA and crude cell lysate as sample material in the sandwich hybridization assay. 18S rRNA was used as target molecule for method evaluation. 5 ml of OD600 = 2 yeast culture was lysed (○) or used for extraction of total RNA (●). Different dilutions of lysed yeast cells or total RNA extracted from the same number of cells were added to the sandwich hybridization solution. Fluorescence (FLU) measured with 18S rRNA probes is presented in relation to the amount yeast cells used for lysis or extraction of RNA. The error bars show the ± SD of three parallel experiments.
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Figure 3: Comparison of extracted total RNA and crude cell lysate as sample material in the sandwich hybridization assay. 18S rRNA was used as target molecule for method evaluation. 5 ml of OD600 = 2 yeast culture was lysed (○) or used for extraction of total RNA (●). Different dilutions of lysed yeast cells or total RNA extracted from the same number of cells were added to the sandwich hybridization solution. Fluorescence (FLU) measured with 18S rRNA probes is presented in relation to the amount yeast cells used for lysis or extraction of RNA. The error bars show the ± SD of three parallel experiments.

Mentions: Fig. 3 shows that when less than 3 × 107 cells as crude lysate were added to the hybridization solution the signal of the respective target molecules was up to 60% higher when compared to signal measured from total RNA extracted from the same amount of cells. When the amount of crude cell lysate was further increased the signal started to decrease probably due to unspecific reactions of the crude cell material. With total RNA the same inhibition effect was observed when more than 2.5 × 108 yeast cells were used for extraction.


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)

Comparison of extracted total RNA and crude cell lysate as sample material in the sandwich hybridization assay. 18S rRNA was used as target molecule for method evaluation. 5 ml of OD600 = 2 yeast culture was lysed (○) or used for extraction of total RNA (●). Different dilutions of lysed yeast cells or total RNA extracted from the same number of cells were added to the sandwich hybridization solution. Fluorescence (FLU) measured with 18S rRNA probes is presented in relation to the amount yeast cells used for lysis or extraction of RNA. The error bars show the ± SD of three parallel experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Comparison of extracted total RNA and crude cell lysate as sample material in the sandwich hybridization assay. 18S rRNA was used as target molecule for method evaluation. 5 ml of OD600 = 2 yeast culture was lysed (○) or used for extraction of total RNA (●). Different dilutions of lysed yeast cells or total RNA extracted from the same number of cells were added to the sandwich hybridization solution. Fluorescence (FLU) measured with 18S rRNA probes is presented in relation to the amount yeast cells used for lysis or extraction of RNA. The error bars show the ± SD of three parallel experiments.
Mentions: Fig. 3 shows that when less than 3 × 107 cells as crude lysate were added to the hybridization solution the signal of the respective target molecules was up to 60% higher when compared to signal measured from total RNA extracted from the same amount of cells. When the amount of crude cell lysate was further increased the signal started to decrease probably due to unspecific reactions of the crude cell material. With total RNA the same inhibition effect was observed when more than 2.5 × 108 yeast cells were used for extraction.

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