ABSTRACT: NOD mice (8- to 13-wk-old) were bred and maintained at the La Trobe University Central Animal House (Bundoora, Melbourne, Australia). All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (National Health and Medical Research Council 1997), after approval by the La Trobe University Animal Ethics committee (Melbourne, Australia). A total of 124 female mice were divided into three groups. The immunized group comprised 91 mice injected s.c. into the lower flanks with 200 micrograms of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Auspep) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco). An i.v. injection of 350 ng of Bordetella pertussis toxin was administered both immediately thereafter and 48 h later. The 26 mice in the control group were injected with adjuvant and pertussis toxin only. The seven animals used for the baseline group (time, day 0) were naive, not injected mice. Except for the seven naive animals from the baseline group, nine animals (seven from the EAE and two from the control group) were sacrificed (at the same time of the day) at each of 13 time points after immunization, which are detailed as days 3, 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, and 19 postimmunization. Lymph nodes from the remaining four animals of the immunized and from the two control groups were dissected, immersed in RNAlater (Ambion), and frozen at 20C. Lymph nodes were removed from RNAlater and homogenized in TRIzol (Invitrogen Life Technologies) using an electric homogenizer. After resuspending the final RNA pellet in water, samples were repurified using the RNeasy kit (Qiagen). cDNA was synthesized from 15 micrograms of total RNA using Superscript II RT (Invitrogen Life Technologies) and a modified dNTP mix containing dUTP. Samples were hydrolyzed by adding 10 microliters of 0.1 N NaOH, neutralized with 25 microliters of 1 M HEPES, and precipitated with 3 M sodium acetate and ethanol. Resuspension in 0.05 M sodium bicarbonate was followed by 1 h incubation with either N-hydroxysuccinimide ester Cy3 or Cy5 fluorescent dyes (Amersham Biosciences). Probes were quenched by the addition of 4 M hydroxylamine and neutralized with 100 mM sodium acetate. Final probe cleanup was conducted using the QIA Quick PCR purification kit (Qiagen). We followed a common reference design in which each Cy3-labeled lymph node probe was combined with a Cy5-labeled probe derived from a pool of RNA. Hybridization onto glass slides containing 18,240 spotted 60- to 70-mer oligonucleotides, followed by washing and scanning was performed at the Gladstone microarray core facility at the University of California (San Francisco, CA) Keywords: Time series
