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BACHD rat samples were provided by CHDI Foundation.
The rat samples were collected under conditions approved by the Danish Agency for Protection of Experimental Animals and by the inhouse Animal Welfare Committee of the Institute for Food and Veterinary Research, DTU.
Rat samples were prepared following the guidelines of the Belgian Regulations for Animal Care.
The miRNAs from human and mouse that were detected in the rat samples were blasted against the rat database in NCBI.
RNA levels for rat samples were measured on Affymetrix RAE230-2 andays and those from mouse samples on Affymetrix MOE430-2 Affymetrixfymetrix, Santa Clara, CA) at The Centre for Applied Genomics (Toronto, Canada).
For DIV, our mouse and rat samples were matched according to standard practice [ 41], but as noted by Valor et al. (2007) [ 34], a rapid change in expression dynamics as a function of DIV may magnify as small mismatch of the species with respect to cell development in culture.
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Brain tissue collected from the rat samples was homogenized in 0.075 M NaCl and 0.024 M ethylenediaminetetraacetic acid (EDTA) buffer, pH 7.5, at a ratio of 1 g of tissue to 1 ml of buffer, and then cooled to 4°C.
Briefly, 2 μg of total RNA from experimental samples and from a common reference rat sample were labelled in separate reactions with Hy3 and Hy5 (Exiqon) fluorescent labels respectively.
For both RT-PCR and Western blot experiments, each human or rat sample was expressed as a ratio to its corresponding β-actin value.
All rats were given diuretics by intragastric administration for 5 days, and the rat urine samples were collected on 1, 3 and 5 days after diuretic administration as described above.
Mouse and rat embryo samples were removed from the data set before identifying tissue-specific NAT probesets for the 9 orthologous tissues in rat and mouse.
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