Location: Forage-animal Production ResearchTitle: Validation of a housekeeping gene for use in bovine vascular gene expression studies) Author
Submitted to: Joint Meeting of the ADSA, AMSA, ASAS and PSA
Publication Type: Abstract only
Publication Acceptance Date: 3/6/2012
Publication Date: 7/15/2012
Citation: Klotz, J.L., Brown, K.R., Matthews, J.C., Boling, J.A., Strickland, J.R. 2012. Validation of a housekeeping gene for use in bovine vascular gene expression studies. J. Anim. Sci. Vol 90, Suppl 3:pg 35. Interpretive Summary:
Technical Abstract: Exposure of ungulate vasculature to ergot alkaloids while grazing endophyte (Neotyphodium coenophialum)-infected tall fescue (Lolium arundinaceum) affects vasoactivity and causes vasoconstriction. Bovine vascular gene expression as affected by exposure to ergot alkaloids in tall fescue is largely unstudied. The objective of this study was to investigate the suitability of ß-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPD), hypoxanthine phosphoribosyl-transferase I (HPRT), succinate dehydrogenase flavoprotein subunit A (SDHA), and ubiquitin C (UBC) as potential housekeeping genes for use in bovine vascular gene expression studies that include different levels of exposure to ergot alkaloids. Lateral saphenous veins (SV) and right ruminal veins (RV) were selected as models for comparison of peripheral and visceral vasculature for future experiments. Veins were collected immediately after slaughter from 19 predominantly Angus steers that had grazed either a low-endophyte-infected tall fescue pasture (LE; 5.7 ha; n=9; BW=266 ±6 kg) or a high-endophyte-infected tall fescue pasture (HE; 5.7 ha; n=10; BW=267 ±6 kg) for 89-105 d. Isolated veins were frozen in liquid N and stored at -80°C until completion of RNA isolation (total) and 1st strand synthesis of cDNA. Real-time PCR was run using SYBR Green with single product verification using dissociation curve. Relative standard curve analysis for each gene was done using serial dilutions of composite cDNA from SV and RV. Melt curve Transcript levels for each gene were analyzed as CRD factorial for endophyte level and vein with mixed models in SAS. None of the 5 genes evaluated were affected by endophyte level or significant for a endophyte level × vein interaction. Levels of HPRT, GAPD, and SDHA were all significant for main effect of vein (P<0.01) caused by a consistently lower expression level in RV tissue (P<0.05). The levels of ACTB and UBC were not different between SV and RV tissues. Thus, when validating microarray data or performing real-time PCR experiments derived from bovine vascular tissue, ACTB or UBC will perform reliably and not be affected by ergot alkaloid exposure of the tissue of origin.