Ty oligonucleotide arrays and studied the dynamics of gene expression in rat duodenum inside 6 h postintrajugular injection of 1,25-(OH)2D3.a-tocopherol, 60 lg menadione, and 40 lg b-carotene in 0.1 ml soybean oil (AEK). Rats were housed in hanging wire cages and maintained on a 12 h light/dark cycle. Rats fed the vitamin D-deficient eating plan have been maintained in a area with incandescent lighting, and all potential sources of ultraviolet light and vitamin D have been excluded. At 14 weeks of age, blood was taken from the tail for measurement of serum calcium concentrations to assess vitamin D depletion. Serum calcium evaluation Blood samples have been obtained in the tail artery. Entire blood was centrifuged at 1100g for 15 min at 25 to yield serum. Serum calcium concentration was determined making use of a 3110 atomic VEGF-D Proteins custom synthesis absorption spectrometer (Perkin lmer, Norwalk, CT) on serum diluted 1:40 with 1 g/L LaCl3 [22]. Experimental design and style Vitamin D-deficient rats have been given intrajugularly one particular dose of 730 ng of 1,25-(OH)2D3/kg of body weight in ethanol or vehicle (for control group) along with a sample of blood was taken quickly just before the injection for serum calcium concentration measurement. Groups of three rats per time point were deeply anesthetized with isoflurane and decapitated at 15 min, 1, 3, and six h right after injection. Blood was collected in the exact same time for determination of alterations in serum calcium concentration. The initial 15 cm of intestine (duodenum) was removed, slit open longitudinally and scraped using the glass slide. The mucosa was homogenized with PowerGen 700 (Fisher Scientific, Pittsburgh, PA) in guanidine thiocyanate (GTC) extraction buffer, supplemented with 2 b-mercaptoethanol (PolyATtract Method 1000, Promega, Madison, WI), flash frozen in liquid N2, and stored at 0 . Experiments have been performed in duplicate. RNA isolation and probe labeling Poly(A)+ RNA was isolated from pooled homogenized mucosa from 3 rats at every single time point. The mRNA was isolated applying the PolyATtract Program 1000 (Promega, Madison, WI) and purified making use of an RNeasy kit (Qiagen, Chatsworth, CA). The good quality, integrity, and quantity from the poly(A)+ RNA was determined by agarose gel electrophoresis, UV absorption spectrophotometry, and Agilent Bioanalyser 2100 (Agilent Technologies, Palo Alto, CA). Microarray probe preparation Double stranded cDNA was synthesized from three lg of polyadenylated poly(A)+ RNA making use of the SuperscriptMaterials and procedures Animals and diets Animals have been maintained and investigation was carried out in accordance with guidelines set forth by the Animal Care and Analysis Committee (University of Wisconsin, Madison, WI). Holtzman male weanling rats were obtained from Sprague awley (Madison, WI) and maintained on a highly purified vitamin D-deficient eating plan, containing 0.47 calcium and 0.three phosphorus (Pi) supplemented three instances per week with 500 lg DL -G.D. Kutuzova, H.F. DeLuca / Archives of Biochemistry and Biophysics 432 (2004) 152Choice system (Invitrogen Life Technologies, Carlsbad, CA), all based on the Affymetrix Gene Expression manual (Affymetrix, Santa Clara, CA). Following phenol/chloroform extraction and ethanol precipitation, a biotin-labeled in vitro transcription reaction was performed applying the cDNA template and IL-17RA Proteins site BioArray Higher Yield In Vitro Transcription kit (Enzo Life Sciences, Farmingdale, NY). The cRNA was fragmented at 0.7 lg/ll final concentration in 1fragmentation buffer (40 mM Tris cetate, pH eight.1, 100 mM potassium acetate, and 30 mM magnesium a.