Black solid flat bottom microplates

To enable year-round comparable data for the years 2007 and 2008, RNA and DNA contents were measured in zooplankton species/stages that were present in all RNAlater-preserved samples; these were copepods Acartia bifilosa females, stages CV–CVI, and rotifers Keratella quadrata (females without eggs). Individual specimens were picked from the samples and transferred to Eppendorf tubes (3 replicate samples per sampling occasion; copepods: 3 ind. sample⁻¹, rotifers: ∼20 ind. sample⁻¹). Nucleic acids were quantified using microplate fluorometric high-range RiboGreen (Molecular Probes, Inc. Eugene, OR) assay after extraction with N-laurylsarcosine followed by RNase digestion [28] , [29] . Fluorescence measurements were done in triplicate for each sample, standard, and negative control using FLUOstar Optima (filters: 485 nm for excitation and 520 nm for emission) reader and black solid flat-bottom microplates (Greiner Bio-One GmbH). Mean standard curve slope ratio (m_DNA/m_RNA) was 1.87.

All enzymatic activities were normalized to the protein concentration in the respective sample. Protein concentrations were determined by a microplate fluorometric assay using the NanoOrange Protein Quantification Kit (Molecular Probes, Inc. Eugene, OR) with bovine serum albumin standards (Jones et al., 2003 (link)). In brief, 10 μl of the homogenate were diluted in NanoOrange working solution to achieve a final volume of 130 μl. Samples were incubated at 95°C for 10 min and cooled to room temperature for 25 min (light protected). Fluorescence was measured with FLUOstar Optima (filters: 485 nm for excitation and 590 nm for emission) reader and black solid flat-bottom microplates (Greiner Bio-One GmbH) with an integration time of 1 s.

We measured RNA:protein ratio and individual protein content as indices of growth and condition in the offspring using the neonates collected during the life table experiment. The animals from each sample were divided between the two assays: 5–15 individuals were used in RiboGreen assay measuring RNA quantity [24 (link)] and 5–10 individuals in NanoOrange assay measuring protein quantity [25 (link)]. Using RiboGreen® Quantitation Kit (Invitrogen, Molecular Probes™), the RNA was quantified after extraction with N-laurylsarcosine followed by RNase digestion [26 ]. Fluorescence measurements were done in triplicate for each sample, standard, and negative control using FLUOstar Optima reader (BMG Labtech, Germany) with 485 nm for excitation and 520 nm for emission and black solid flat-bottom microplates (Greiner Bio-One GmbH). For protein measurements with NanoOrange® Protein Quantitation Kit (Invitrogen, Molecular Probes™), Bovine serum albumin (BSA, Molecular Probes) was used as a protein standard. The samples were analysed according to the manufacturer guidelines using the same type of microplates, plate reader and wavelengths as for the RNA quantification.