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Peripheral blood samples were collected from every subject evaluated; each sample was assessed for the presence of HPV-DNA as described below.
Since we attempted to obtain as global a view as possible of the post-T/HS inflammatory response, serum samples were collected at the end of every time point and each sample was assessed for basic FGF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α, TNF-α, and basic VEGF using a mouse-specific bead set (Table S1).
Each sample was assessed in duplicate and every experiment was performed at least 3 times.
Cell number and viability for each sample was assessed using 0.4% Trypan blue solution and a hemocytometer.
Quality of the sequenced reads for each individual sample was assessed using the FastQC program (see Code availability 1).
Quality and quantity of the Total RNA sample was assessed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies; Santa Clara, CA, USA).
The LCBD represents the ecological comparative uniqueness of the sample, and its significance for each sample was assessed through a permutation analysis (p < 0.05, 999 iterations) testing the null hypothesis that species' distribution is random among sampling units.
Each sample was assessed by cytologic examination and RT-PCR analysis for tumor markers: CEA, CK7, Kras2, and MUC1.
The overall bracket failure rate of the whole sample was assessed.
The integrity of the RNA sample was assessed by agarose gel electrophoresis.
The purity of the sample was assessed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. S1).
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