ABSTRACT: Female C57BL/6J mice were fed a vitamin D deficient diet (0.47% calcium, 0.3% phosphate; Teklad diet TD 89123, Envigo Teklad diets, Madison, Wisconsin) 2 -3 weeks prior to mating to a male C57BL/6J male mouse and during subsequent pregnancy and lactation. Pups from these mice were maintained on this diet until 12-14 weeks of age. For this study, mice were maintained in a virus and parasite-free barrier facility and exposed to a 12h-light, 12h-dark cycle. Food and water were given ad libitum. All the animal experiments conducted were approved by the Rutgers, New Jersey Medical School Animal Care and Use Committee. Mice were ordered from Charles River Laboratories. 12 – 14 week old vitamin D deficient mice were randomly separated and injected with either 0.1 ml vehicle (9:1 mix of propylene glycol and ethanol) or 1,25(OH)2D3 (from Cayman Chemical Company Ann Arbor, MI) (ip 10ng/g body weight) and sacrificed 4h after the injection. Intestinal tissues were harvested from mice. About 10-15 cm of the proximal half of small intestine were used for the duodenum crypts and villi samples, and the entire colon tissues from the terminal cecum to rectum were used for colon samples. After flushing with cold PBS, the intestines were cut opened longitudinally, cut into 1 cm pieces in cold PBS for washing, and then incubated with 3mM EDTA/PBS in rotator for 5 min at 4 0C. The solution was discarded and the tissues were incubated with fresh 3mM EDTA/PBS in rotator for 10 min at 4 0C. After shaking the tubes gently for 10 times, the intestinal pieces were transferred to new tube prepared with cold 3mM EDTA/PBS, rotated for 30 min at 4 0C, then were shaken 30 times. The supernatant containing the whole epithelium tissues (crypts and villi) were collected and spun down at 200 rcf for 3 min at 4 0C. The pellet was resuspended with cold PBS and villi separated from crypts with 70 µm filter. Colon tissue was scraped with a glass slide to remove the epithelial mucosa and washed with PBS. All samples were centrifuged tubes at 200 rcf for 3 mins at 4 0C and at 300 rcf for 30 seconds at 40C in order to remove any residual PBS. We then proceeded to RNA extraction. RNeasy Plus Universal Kit was used for villi and crypts with RiboZol RNA extraction reagent (Amresco, Solon, Ohio), according to manufacturer’s instructions. All nucleic acid extracts were gDNA Eliminator Solution for 15 s at 37 °C, in order to remove contaminating chromosomal DNA. Resulting RNA was analyzed for quantity and quality with a NanoDrop spectrophotometer ND-1000 (Isogen Life Science, Utrecht, The Netherlands).
