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We hypothesized that a metabolomics approach would be able to stratify HCV related liver histological disease progression by analysis of patient sera samples using a quantitative NMR metabolomics profiling approach.
Thus, in line with previous steroid-abuse rat studies [36 39], the present study was designed with a daily dose of 5.0 mg/kg for 6 consecutive days, followed by analysing hair, urine and sera samples using newly developed LC-MS/MS methods.
Analysis of the human sera samples using GC-TOF-MS yielded 144 known metabolic structures and a total of 354 molecular features (Dataset).
Together, these validations showed that the method is valid for the quantitation of human sera samples using calibration curves in serum, as well as using calibration curves in plasma (Tandem Labs 1999, 2001a, 2001b).
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Then, we isolated the total RNA from each pooled sera sample using TRIzol reagent (Invitrogen) according to the manufacturer's instructions with minor modifications.
When the generalised and combinational ROC curves were applied to the samples using sera from the healthy women as the controls these curves were not as informative as some of the individual features (see Figure 4B) but in these latter cases, only a small number of sera were positive (see Figure 3A).
Poly(A -enriched mRNA -enrichedied fromRNAe total RNA samples using Sera-mega Oligo(dT) beads (Illumina) and fragmented wash divalent cations at elevated tempurified.
Poly (A) mRNA was enriched from 20 μg of total RNA from the NaCl-treated pear samples, using Sera-mag Magnetic Oligo (dT) beads (Thermo Fisher Scientific, USA).
Using the reagents of the mRNA Sample Prep Kit and in accordance with the RNA sequencing protocol provided by Illumina, poly(A)+ mRNA was selected out from the total RNA samples using Sera-mag magnetic oligo dT) beads.
Viral DNA was extracted from 200 μL sera per sample using an AccuPrep Genomic DNA Extraction Kit (Bioneer, Korea) or QIAamp MinElute Virus Spin (Qiagen, Germany) as instructed in the manufacturer's manuals.
The O.D. value of the age-matched naïve sera was subtracted from the samples using antisera from vaccinated mice.
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