The two kidneys from each animal were cut in half longitudinally; one half was put in 10% neutral buffered formalin for histology, and the other 3 halves were snap frozen in liquid nitrogen and stored at -80°C until further use.
Growth protocol
Male Wistar rats were separated in two groups: The control group received a standard rodent diet (Specialty Foods, Perth, Australia) and tap water ad libitum for 6 months. The experimental group was fed 60 mM lithium/kg dry food for 6 months. Water was available ad libitum. All rats given lithium were supplied with a salt block to maintain sodium balance and prevent lithium intoxication. At 6 months, the animals were sacrificed by decapitation and their kidneys removed.
Extracted molecule
total RNA
Extraction protocol
Total RNA from the kidneys was isolated with TrizolR (Invitrogen, Life Technologies, USA), followed by cleaning in Norgen Total RNA columns (Norgen Biotek Corp., Canada). RNA was quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and its (purity and) quality determined on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
50 ng total RNA was processed by using a GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA) and hybridised to Affymetrix Rat Gene 1.0 ST gene chips.
Scan protocol
Chips were stained and washed with an Affymetrix GeneChip Fluidics Station 450, and scanned in an Affymetrix GeneChip 7G Plus scanner. All procedures were done according to the manufacturer's instructions.
Data processing
Cel files were imported with RMA background correction, quantile normalization, log base 2 transformation, and median polish, using PArtek Genomcis Suite v 6.5 (www.Partek.com). Batch effect removal was applied using ANOVA for the factor Scan date. Gene summary and quantification was performed on the basis of the mean of the expression values of the exons per gene. probe group file: RaEx-1_0-st-v1.r2.pgf meta-probeset file: RaEx-1_0-stv1.r2.dt1.rn4.full.mps
