Abstract:

Monitor cytokine expression for assessment of normal or aberrant immune system function.

Suggested Citation:

Wetzel, Dana, Erin Pulster, and Kristina Deak. 2014. Immune Function Assays of Sharks, northeastern Gulf of Mexico and West Florida Coast, 2010-2011. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7FN144T

Purpose:

Monitor cytokine expression for assessment of normal or aberrant immune system function.

Data Parameters and Units:

Once antibody-based assay is developed, ideally could be measured in ng/mL. Wetzel_cytokines.xlsx-- Sample Info: MML #, Sample ID, Catch ID, matrix (liver, muscle, blood, placenta, whole pup, gonads, kidney, serum), analysis (PAH, comet, fertility, immune), Set ID, Collection Date (MM/DD/YYYY), Time (HH:MM), Common name (Silky, Shortfin Mako, Wahoo, Scalloped hammerhead, Oilfish, Pelagic stingray, Yellowfin tuna, Escolar, Mahi Mahi, Swordfish, Sandbar, Spinner, Blacktip, Baracuda, Dusky, Bull, Blacknose), Species, Sex (Male, Female, pup), PCL Precaudal Length (cm), FL Fork Length(cm), TL Total Length(cm), STL Stretch Total Length (cm), Girth, Weight(kg), Liver wt (kg), Maturity (Mature, Immature, pup), Comments. Cytokines: Catch ID, Species (Sandbar, Silky, Bull, Tiger), IFNy Conc. (pg/mL), IL-10 Conc. (pg/mL), IL-1 Conc. (pg/mL), TNFa Conc. (pg/mL)

Methods:

Serum samples collected in 2010-11 will be screened with an antibody-based assay, once it has been developed based upon research performed with spleen and whole blood samples.

Instruments:

BioRad MyCycler Thermocylcer for reverse transcription of RNA to cDNA and PCR. Gel electrophoresis unit and chemidoc for running agarose gels to confirm gel band sizes. Nanodrop spectrophotometer to confirm quality and purity of RNA isolated from samples. Real time PCR instrument with software to quantify fold change in gene expression. BioPlex 3D with xMAP technology for creating multiplex panel with species-specific antibodies.

Error Analysis:

For the genetics work, sample blanks and reverse transcription blanks are included in each run to confirm that no genomic DNA contamination has occurred in the RNA isolation process. Beta actin is included in PCR runs as the house-keeping gene. For the antibody assay, %CV for the standard curve less than 25% is acceptable, less than 15% for sample replicates. The Bioplex 3D instrument is calibrated before each use.

Individual Files: