Mice were infected intravenously, through a lateral tail vein, with 106 CFU of M. avium. Control animals received the same volume of saline. At the time points of interest, mice were sacrificed and blood and tissues were harvested for the different analysis. For bacterial quantification, the livers and spleens were aseptically collected and homogenized in a 0.05% Tween 80 solution in distilled water. Serial dilutions were plated into Middlebrook 7H10 agar medium and the plates were incubated at 37 ºC for 1 week, when the colonies were counted To induce iron overload, mice were injected intraperitoneally with 10 mg of iron, as iron-dextran (Sigma). Control mice received an equivalent amount of dextran (Sigma) by the same route. Animals were infected two weeks after the iron-dextran treatment. Iron overload was confirmed by quantifying liver and spleen non-heme iron by the bathophenanthroline method as previously described (Rodrigues et al., 2006)
Growth protocol
The BALB/c (NRAMP1-S) and C.D2 (NRAMP1-R) congenic mouse strains were bred and housed at the Instituto de Biologia Molecular e Celular (IBMC) animal facility. The animals were kept inside individually ventilated cages, bearing high efficiency particulate air (HEPA) filters and were fed sterilized food and water ad libitum. For the experimental treatments, 8-12 weeks old females were used. All animal experiments were carried out in compliance with the animal ethics guidelines of the institute, and the national and European regulations for the care and handling of laboratory animals Mycobacterium avium strain 2447, forming smooth transparent (SmT) colonies, was isolated from an AIDS patient and was a gift from Dr. F. Portaels (Institute of Tropical Medicine, Antwerp, Belgium). Mycobacteria were grown to mid-log phase in Middlebrook 7H9 medium (Difco, Sparks, MD) containing 0.05% Tween 80 (Sigma, St. Louis, MO) at 37ºC. Bacteria were harvested by centrifugation, suspended in a small volume of saline containing 0.05% Tween 80, briefly sonicated to disrupt bacterial clumps, diluted and stored in aliquots at -70ºC until use
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from snap frozen liver samples using the Qiagen (Hilden, Gerrmany) Rneasy Midi Kit according to the manufactur's specifications.
Label
Cy3
Label protocol
Fluorescent cDNA probes were synthesized from 5 µg total RNA using a linear mRNA amplification protocol, exactly as described in (http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html). 3µg of the T7 RNA polymerase amplified antisense RNA was subsequently subjected to a direct labeling reaction by incorporation of Cy3 fluorescent dyes using random primers
Mice were infected intravenously, through a lateral tail vein, with 106 CFU of M. avium. Control animals received the same volume of saline. At the time points of interest, mice were sacrificed and blood and tissues were harvested for the different analysis. For bacterial quantification, the livers and spleens were aseptically collected and homogenized in a 0.05% Tween 80 solution in distilled water. Serial dilutions were plated into Middlebrook 7H10 agar medium and the plates were incubated at 37 ºC for 1 week, when the colonies were counted To induce iron overload, mice were injected intraperitoneally with 10 mg of iron, as iron-dextran (Sigma). Control mice received an equivalent amount of dextran (Sigma) by the same route. Animals were infected two weeks after the iron-dextran treatment. Iron overload was confirmed by quantifying liver and spleen non-heme iron by the bathophenanthroline method as previously described (Rodrigues et al., 2006)
Growth protocol
The BALB/c (NRAMP1-S) and C.D2 (NRAMP1-R) congenic mouse strains were bred and housed at the Instituto de Biologia Molecular e Celular (IBMC) animal facility. The animals were kept inside individually ventilated cages, bearing high efficiency particulate air (HEPA) filters and were fed sterilized food and water ad libitum. For the experimental treatments, 8-12 weeks old females were used. All animal experiments were carried out in compliance with the animal ethics guidelines of the institute, and the national and European regulations for the care and handling of laboratory animals Mycobacterium avium strain 2447, forming smooth transparent (SmT) colonies, was isolated from an AIDS patient and was a gift from Dr. F. Portaels (Institute of Tropical Medicine, Antwerp, Belgium). Mycobacteria were grown to mid-log phase in Middlebrook 7H9 medium (Difco, Sparks, MD) containing 0.05% Tween 80 (Sigma, St. Louis, MO) at 37ºC. Bacteria were harvested by centrifugation, suspended in a small volume of saline containing 0.05% Tween 80, briefly sonicated to disrupt bacterial clumps, diluted and stored in aliquots at -70ºC until use
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from snap frozen liver samples using the Qiagen (Hilden, Gerrmany) Rneasy Midi Kit according to the manufactur's specifications.
Label
Cy5
Label protocol
Fluorescent cDNA probes were synthesized from 5 µg total RNA using a linear mRNA amplification protocol, exactly as described in (http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html). 3µg of the T7 RNA polymerase amplified antisense RNA was subsequently subjected to a direct labeling reaction by incorporation of Cy3 fluorescent dyes using random primers
Hybridization protocol
The microarrays were immersed at 42°C in 6xSSC/0,5%SDS/1%BSA for 40 min and subsequently washed extensively with ddH2O at room temperature. Prior to hybridization, the spotted PCR products were denatured by immersing the slides at 95°C in ddH2O for 2 min. Excess of liquid was removed from the slides by centrifuging them briefly at 715xg in a microtiter plate centrifuge (Z320, Hermle, Wehingen, Germany). Prior to hybridization, the purified Cy3 and Cy5 labeled cDNAs were mixed, 5 µg poly(dA) and 1 µg human Cot1 DNA (both Gibco Invitrogen Corp., Carlsbad, Ca, USA) were added and subsequently evaporated in a vacuum Concentrator 5301 (Eppendorf, Hamburg, Germany) at 60°C. The resulting pellet was dissolved in 12 µl hybridization buffer (50% formamide / 6xSSC / 0,5%SDS / 5x Denhardt) and denatured by incubating at 95°C for 2 min. The probe was then transferred onto the array under a 24x24 mm coverslip and incubated in a humid chamber (GeneMachines, San Carlos, CA, USA) containing 2xSSC drops for providing humidity. Hybridization was performed for 12h to 16h in a 42°C waterbath (GFL, Burgwedel, Germany). After hybridization the microarrays were washed in 0,1xSSC / 0,1%SDS for 10 min and twice with 0,1xSSC for 5 min (on an orbital shaker), followed by a brief immersion of the slides in ddH2O. Finally, the washed slides were dried by centrifuging them briefly at 715xg in a microtiter plate centrifuge (Z320, Hermle, Wehingen, Germany). All washing steps were performed at room temperature.
Scan protocol
All microarrays were scanned on a GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA). For each microarray individual laser power and photomultiplier settings were used, allowing all signals to remain in the linear range of the scanner. Separate scan images for Cy3 and Cy5 were produced
Description
pool of five mice each
Data processing
Intensity values for each spot were calculated by subtraction of the local background surrounding the spot. All spots were used for the calculation of a linear regression line. The regression line’s parameters (offset and slope) were used for normalization. The data evaluation performed using custom bioinformatic toll called Iron Chip Evealuation package (ICEP). “ICEP” is a software package designed specifically for “iron chip” data analyzing. This package is able to use all advantages of the Iron Chip microarray design.The values in the Sample data tables have been processed in a dyeswap aware fashion so the ratio presented for the first direction hybridization has the ratio cy3/cy5 whereas the 2nd direction hyb is the cy5/cy3 ratio accounting for the dyeswap.
