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Lanthanum chloride (0.5%, w/v) was added to both samples and standard solutions to avoid chemical interferences, as referred by Kawashima and Valente-Soares [25].
Both samples and standard were placed in a water bath, heated for 45 min at 90°C, ice-cooled to stop the reaction, and then extracted once with 250 μL of n-butanol (Sigma).
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The buffer (TISAB-III) solution was added to both samples and standards.
Enzyme activity, defined as percent inhibition of WST-1 reduction, was determined as {[(reagent control vo−buffer blank vo)−(sample vo−sample blank vo)]/(reagent control vo−buffer blank vo)}×100 for both samples and standards.
They are purposely added to both samples and standards at the same concentration in order to provide a basis for comparison in quantification.
Iron reducing agent (5 μl) was added to both samples and standards (0 10 nmol/well in a total volume of 100 μl) followed by incubation at room temperature for 30 min.
Both sample and standard stock solutions were stored at 2 8°C protected from light.
At least 1300 nuclei in both sample and standard G1 peaks were analysed per sample (Suda et al. 2007).
Briefly, both unknown samples and standard curve samples made from HUVEC RNA dilutions of known concentration ranging from 500 ng–1 pg RNA were made up to 12 µl volume using RNase-free water.
The injection of both extracts from samples and standard solutions (1 μL) was performed by hand.
Specificity was established by lack of interfering peaks at the retention time for 11 mins for both sample and the standard and 8(b)).
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