Dr. Roberta Brambilla and Dr. John Bethea, The Miami Project to Cure Paralysis, Miller School of Medicine, University of Miami
Treatment protocol
Mice were immunized with MOG(35-55) peptide to induce EAE, as previously described (2). MOG peptide was synthesized by standard 9-fluorenyl-methoxycarbonyl chemistry and was >95% pure as determined by reversed-phase-HPLC (Biosynthesis International, Lewisville, TX). Mice were injected subcutaneously on days 0 and 7 with 150 ug of MOG peptide emulsified in complete Freund’s adjuvant (DIFCO Laboratories, Detroit, MI). In addition, on days 0 and 2 post immunization (p.i.), mice were given pertussis toxin (500 ng/mouse, Sigma-Aldrich, St. Louis, MO) by intra-peritoneal injection. Mice were sacrificed 10, 17 or 80 days after induction of EAE.
Growth protocol
Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Miami. All mice used were obtained by breeding heterozygous GFAP-IkappaBalpha-dn males with WT females. WT littermates were used as controls. Animals were housed in a 12 hr light/dark cycle in a virus/antigen free facility with controlled temperature and humidity and provided with water and food ad libitum.
Extracted molecule
total RNA
Extraction protocol
To isolate total RNA from the samples, 1 mL of TRI Reagent (Molecular Research Center Cat #TRI-118) was added per 5 X 106 cells. The sample was then macerated using a Kinematica AG Polytron PT-2100 for 1 min. After a 5 min incubation at room temperature 0.2 volumes of chloroform was added and mixed, following by a 3 min incubation at room temperature and 10 min centrifugation at 12,000 g 4°C. The top aqueous layer was transferred to a new tube containing 0.5 volume of isopropanol and 2 to 10 ul of polyacryl carrier (Molecular Recearch Center, Cat #PC152). After mixing, the precipitate containing the total RNA was collected by 15 min of centrifugation at 12,000 g 4°C. The pellet was then washed two times with 70% ethanol. Total RNA was additionally purified with RNeasy Mini Kit (Qiagen, Cat # 74106). Total RNA yield was determined spectrophotometrically.
Label
Cy5
Label protocol
1ug of total RNA was amplified and labeled using Amino Allyl MessageAmp aRNA Amplification Kit (Ambion, Cat#1753) and Cy3- and Cy5-NHS ethers (Amersham, Cat # PA23001 and PA25001, respectively).
Mice were immunized with MOG(35-55) peptide to induce EAE, as previously described (2). MOG peptide was synthesized by standard 9-fluorenyl-methoxycarbonyl chemistry and was >95% pure as determined by reversed-phase-HPLC (Biosynthesis International, Lewisville, TX). Mice were injected subcutaneously on days 0 and 7 with 150 ug of MOG peptide emulsified in complete Freund’s adjuvant (DIFCO Laboratories, Detroit, MI). In addition, on days 0 and 2 post immunization (p.i.), mice were given pertussis toxin (500 ng/mouse, Sigma-Aldrich, St. Louis, MO) by intra-peritoneal injection. Mice were sacrificed 10, 17 or 80 days after induction of EAE.
Growth protocol
Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Miami. All mice used were obtained by breeding heterozygous GFAP-IkappaBalpha-dn males with WT females. WT littermates were used as controls. Animals were housed in a 12 hr light/dark cycle in a virus/antigen free facility with controlled temperature and humidity and provided with water and food ad libitum.
Extracted molecule
total RNA
Extraction protocol
To isolate total RNA from the samples, 1 mL of TRI Reagent (Molecular Research Center Cat #TRI-118) was added per 5 X 106 cells. The sample was then macerated using a Kinematica AG Polytron PT-2100 for 1 min. After a 5 min incubation at room temperature 0.2 volumes of chloroform was added and mixed, following by a 3 min incubation at room temperature and 10 min centrifugation at 12,000 g 4°C. The top aqueous layer was transferred to a new tube containing 0.5 volume of isopropanol and 2 to 10 ul of polyacryl carrier (Molecular Recearch Center, Cat #PC152). After mixing, the precipitate containing the total RNA was collected by 15 min of centrifugation at 12,000 g 4°C. The pellet was then washed two times with 70% ethanol. Total RNA was additionally purified with RNeasy Mini Kit (Qiagen, Cat # 74106). Total RNA yield was determined spectrophotometrically.
Label
Cy3
Label protocol
1ug of total RNA was amplified and labeled using Amino Allyl MessageAmp aRNA Amplification Kit (Ambion, Cat#1753) and Cy3- and Cy5-NHS ethers (Amersham, Cat # PA23001 and PA25001, respectively).
Hybridization protocol
Equal amounts of labeled aRNA were hybridized to Agilent Whole Mouse Genome Oligo microarrays for 17 hours at 65°C according to manufacturer's instructions.
Scan protocol
The microarrays were scanned at 5 micron resolution using a GenePix 4100A scanner (Axon Instruments at Molecular Devices) and the resulting images were analyzed with the software package GenePix Pro 5.1 (Axon Instruments at Molecular Devices).
Description
1. Brambilla, R., V. Bracchi-Ricard, W.H. Hu, B. Frydel, A. Bramwell, S. Karmally, E.J. Green, and J.R. Bethea. 2005. Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J Exp Med 202:145-156. 2. Szalai, A.J., S. Nataf, X.Z. Hu, and S.R. Barnum. 2002. Experimental allergic encephalomyelitis is inhibited in transgenic mice expressing human C-reactive protein. J Immunol 168:5792-5797.
Data processing
Data extracted from the images were transferred to the software package Acuity 4.0 (Axon Instruments) for normalization and statistical analysis. Each array was normalized for signal intensities across the whole array and locally, using Lowess normalization. Features for further analysis were selected according to the following quality criteria: (1) at least 90% of the pixels in the spot had intensity higher than background plus two standard deviations; (2), there were less than 2% saturated pixels in the spot; (3) signal to noise ratio (defined as ratio of the background subtracted mean pixel intensity to standard deviation of background) was 3 or above for each channel; (4) the spot diameter was between 45 and 75 micron; (5) the regression coefficient of ratios of pixel intensity was 0.6 or above.
