Goal of experimet: Identification of differentially expressed immune genes from male and female BWF1 lupus-prone mice. (Female incidence is higher than male--attempting to find sex hormone regulated genes that may contribute to this difference)
Whole spleen was taken from a pre-lupus (4 mo. old) (New Zealand Black x New Zealand White)F1 (BWF1) mouse.
Preparation of cDNA. Double-stranded cDNA was synthesized from purified RNA. The first strand was synthesized by incubating 5 μg of RNA with 100 pg/ml T7-(dT)24 primer (HPLC purified DNA primer sequence: 5’-GGCCAGTGAATTGTAATACG ACTCACTATAGGGAGGCGG-(dT)24 -3’ Genset Corp, San Diego, CA) at 70°C for 10 minutes. Samples were incubated for 1 hour at 42°C with the following mix: 1X first strand buffer, 10 mM dithiothreitol, 500 μM each dNTP, 200 U SuperScript II in diethylpyrocarbonate (DEPC)-treated water up to 20 μl. Second strand synthesis was performed by incubating the first strand with the following mix for 2 hours at 16°C: 1X second strand reaction buffer, 200 μM dntps, 10 U E. coli DNA ligase, 40 U E coli DNA Polymerase I, 2 U of E. coli RNase H up to 150 μl with DEPC-treated water (all reagents were contained in SuperScript Choice System for cDNA Synthesis, Invitrogen). A phenol/chloroform extraction was performed on the ds-cDNA preparation before biotin-labeled cRNA was generated. Synthesis and fragmentation of biotin-labeled cRNA (in vitro transcription). The ENZO BioArrayTM HighYieldTM RNA Transcript Labeling Kit (T7) (Enzo diagnostics, Inc., Farmingdale, NY) was used to produce large amounts of hybridizable biotin-labeled RNA targets by in vitro transcription from the ds-cDNA. The following mix was incubated at 37°C for 5 hours: 1 μg of ds-cDNA, 1X HY reaction buffer, 1X biotin labeled ribonucleotides, 1X dithiothreitol, 1X T7 RNA Polymerase. Biotin-labeled cRNA was run over RNeasy spin columns (Qiagen), quantified, and run on an agarose gel to visualize the size distribution of labeled transcripts. Twenty micrograms of cRNA was incubated with 1X fragmentation buffer for 35 minutes at 94°C. (5X fragmentation buffer: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc). After fragmentation, the samples were stored at -20°C until the hybridization was performed. Sample hybridization. Oligonucleotide microarrays (MGU74v2 A, B, and C GeneChip probe arrays; Affymetrix) were hybridized with labeled cRNA derived from spleens from individual mice. For each array,15 μg of fragmented cRNA was mixed with a hybridization cocktail consisting of 1X hybridization buffer (2X hybridization buffer: 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween), 0.5 mg/ml acetylated BSA (Invitrogen), 0.1 mg/ml herring sperm DNA (Promega), and water (BioWhittaker) up to 300 μl). Biotin labeled cRNA transcripts of the E. coli and P1 bacteriophage genes, BioB, bioC, bioD, and cre (GeneChip Eukaryotic Hybridization control kit, Affymetrix) were spiked into each hybridization mix at 1.5, 5, 25, and 100 pM to evaluate sample hybridization efficiency for each array. The hybridization cocktail was heated to 99°C and then 45°C for 5 minutes each before it was centrifuged to remove any insoluble material. The array was equilibrated to room temperature, moistened with 1X hybridization buffer, and incubated for 10 minutes at 45°C with rotation. After incubation, the buffer solution was removed from the array. The array was filled with 300 μl of the hybridization cocktail, placed in a rotisserie box in a 45°C oven, and incubated for 16 hours while rotating at 60 rpm. Washing and staining of array. The hybridization cocktail was removed and the GeneChip Fluidics Station 400 (Affymetrix) with Microarray Suite software (Affymetrix) was used to wash and stain the probe arrays with the following protocol: 10 cycles of 2 mixes/cycle with wash buffer A at 25°C, 4 cycles of 15 mixes/cycle with wash buffer B at 50°C, 30 minute incubation with staining solution at 25°C, 10 cycles of 4 mixes/cycle with wash buffer A at 25°C. Wash buffer A -- non-stringent wash buffer (6X sodium chloride sodium phosphate + ethylenediaminetetraacetic acid (SSPE), 0.01% Tween-20). (20X SSPE: 3 M NaCl, 0.2 M NaH2PO4, 0.02 EDTA) (BioWhittaker). Wash buffer B – stringent wash buffer (100mM MES, 0.1 M [Na+], 0.1% Tween 20). Staining solution (1X 2-(N-Morpholino)ethanesulfonic Acid (MES) stain buffer, 2 mg/ml acetylated BSA, 10 μg/ml Streptavidin Phycoerythrin (SAPE), and water up to 600 μl). (12X MES stain buffer: 1.22 M MES, 0.89 M [Na+]). Analysis. After staining, the probe arrays were scanned using the GeneChip 3000 Scanner (Affymetrix) with Microarray Suite software (Affymetrix). Technical and assay variation between arrays was corrected for by multiplying or dividing the overall intensity of each array by a scaling factor so that the overall intensity of each array was equivalent to facilitate comparison analysis. Keywords = disease-prone (female) vs not disease prone (male)
