ABSTRACT: Male weanling Wistar rats from the Animal Facility at the Center for Experimental and Applied Pathology were divided into 4 groups and fed the following diets: 1) choline-deficient diet with VO [corn and hydrogenated oils) as lipids (CDVO); 2) choline-supplemented diet with VO as lipids (CSVO); 3) choline-deficient diet with MO as lipid (CDMO); and 4) choline-supplemented diet with MO as lipid (CSMO). Authors have adhered to appropriate NIH Guide for the Care and Use of Laboratory Animals. It is known that female rats are more resistant than male rats to AKI. Animals were sacrificed after receiving the experimental diets for 6 days. The left kidney was fixed in formaldehyde-buffer and stained with hematoxiline-eosin for histopathological analysis. The right kidney was cryopreserved for microarray analysis. Cryopreserved kidney was wrapped with aluminum foil and broken with a hammer previously wrapped with tape paper on a counter covered in aluminum. The pieces of the kidney were located in a mortar with liquid nitrogen to keep cryopreservation and were pulverized with a pestle. Nitrogen was added as it evaporated. The tissue was broken up to be completely pulverized. Powder was placed with a spatula in a cryotube supported on a dry ice with a layer of aluminum above. Before proceeding with another sample and to avoid contamination, the mortar, the pestle and the spatula were washed with tap water, distilled water and then alcohol. The tape of the hammer, the aluminum on the counter and the latex gloves were also replaced by new ones. Total RNA was purified from 30 milligrams of frozen rat kidney pools, using RNeasy Mini Kit [Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The biological concentration, integrity and quality of the RNA obtained were performing using NanoDrop 2000c (Thermo Fisher Scientific, Delaware, USA) and RIN (RNA Integrity Number). Five hundred nanograms of total RNA were processed and hybridized to Affymetrix GeneChip Rat Gene 1.0 ST Array (Affymetrix Inc, Singapore, Singapore), according to Ambion WT Expression Kit instructions (Ambion Inc, Texas, USA). Total RNA obtained during the tissue extraction was processed to obtain a double strand cDNA. After that we performed a in-vitro transcripition to generate antisence cRNA (aRNA). This aRNA was used to generate a single-stranded DNA (ss-DNA) using random primers and the dUTP +dNTP mix. The resulting single-stranded DNA (ss-DNA) containing the unnatural uracilbase is then treated with Uracil DNA Glycosylase, which specifically removes the uracilresidue from the ss-DNA molecules. In the same reaction, the APE 1 enzyme then cleaves the phosphodiester backbone where the base is missing, leaving a 3’-hydroxyland a 5’-deoxyribose phosphate terminus. Before this prosses, shorts ss-DNA fragments were labeled by terminal deoxynucleotidyl transferase (TdT) that covalently linked the 3’-hydrosyl phosphate terminus whit Biotin Allonamide Triphosphate. The GeneChip® Rat Gene 1.0 ST Array enables whole-genome, gene-level expression studies for well-characterized genes. It is a single GeneChip®-brand array comprised of more than 722 254 unique 25-mer oligonucleotide features accounting for more than 27 342 gene-level probe sets. Results were scanned with GeneChip® Scanner 3000 7G (Affymetrix Inc, Tokyo, Japan), and normalized by RMA algorithm using Affymetrix Expression Console Software. In addition, call values were retrieved by MAS5 algorithm, and only genes with a “p” (present) call value were used in the analysis. Differentially expressed genes were identified using limma (www.bioconductor.org) and “p” adjusted values and absolute log fold change greater than 1.5 were used for gene selection.
