strain: C57Bl/6 Sex: Male diet: DHA diet-fed tissue: lung
Treatment protocol
We employed a previously established murine model that utilizes an intranasal exposure of HDE to induce airway inflammation for our in vivo investigations [30,31]. Studies were performed as 3 independent experimental sets, for total of 8 mice for saline-treated groups and 12 mice for HDE-treated groups. Mice were lightly anesthetized by isoflurane inhalation prior to receiving a single intranasal challenge of 50 μL sterile saline (PBS) or 12.5% HDE. Four weeks prior to receiving the intranasal instillation, mice were initiated on either a DHA-rich diet or control diet containing no DHA. The animal chow diets were prepared by Envigo (Madison, WI) using a base AIN-93G diet. The DHA diet preparation was the same as previously described [30,33]. Briefly, to prepare the DHA-rich diet, soybean oil from the AIN-93G base diet was replaced with DHASCO oil (DSM Nutritional Products, Kingstree, SC), a DHA-based oil containing 39.2% DHA and high-oleic safflower oil. The control diet, containing no DHA, modified the same AIN-93G diet by replacing soybean oil with high-oleic safflower oil. The newly formulated DHA-rich diet provides the mice with approximately 1.4% of total caloric intake strictly from DHA. Five hr following the single HDE intranasal challenge, mice were euthanized.
Growth protocol
Male wildtype C57BL/6J mice 5–8 weeks of age were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the University of Nebraska Medical Center (UNMC) Comparative Medicine Facilities. Mice were allowed free access to food and water and housed in micro-isolator cages (five per cage). The food was changed weekly by the investigators while the water was changed by UNMC animal care staff. Each mouse was weighed weekly (see Supplemental Figure 1 for initial and final weights) and examined for any signs of distress. All related experiments and procedures were approved by the UNMC Institutional Animal Care and Use Committee.
Extracted molecule
total RNA
Extraction protocol
The left lungs were collected and stored in RNA later (ThermoFisher, Waltham, MA). Randomly selected left lung tissues (n=24) out of the possible 40 mice, representing tissues collected across three unique experimental trials, were homogenized and RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). RNA sample quality was quantified using NanoDrop ND-100 (NanoDrop Technologies, Inc, Wilmington, DE) and an Agilent 2100 Bioanalyzer (UC Riverside Core Facilities, Agilent Technologies, Santa Clara, CA).
Label
n/a
Label protocol
Assessment of transcript-level gene expression changes was performed using the NanoString mouse Immunology Panel (NanoString Technologies, Seattle, WA, USA), a codeset designed to target 561 genes related to inflammation and immune response. Fifty to 100 ng of total RNA was mixed with the codeset and reporter probes and hybridized for 16 hr to form a purified target-probe complex that was then imaged and quantified by a nCounter Sprint profiler.
Hybridization protocol
n/a
Scan protocol
Chip was imaged and quantified by a nCounter Sprint profiler.
Description
codeset and reporter probes for Nanostring platform GPL19964
Data processing
Gene expression data analysis was performed using the nCounter Analysis System, nSolver 4.0 software. The expression data were normalized by using a geometric mean of 4 housekeeping genes: OAZ1, PPIA, RPL19, and EEF1G. Following normalization, 20 of the samples passed normalization and were viable to use for the subsequent analyses, including 4 Control Diet + saline, 6 Control diet + HDE, 3 DHA diet + saline, and 7 DHA + HDE samples.
