title: Differentiated Subcutaneous Replicate 2B

description:
Trizol isolation of total RNA from Differentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.100000000000001, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 9.9030000000000005, Corner + 111, Corner -  111, Background 69.72, Noise 3.74, Raw Q 3.0600000000000001
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Omental Replicate 2B

status: Public on Jun 01 2005

type: RNA

city: Cambridge

first: Garrett

middle: M

last: Frampton

state: MA

address: 9 Cambridge Center

department: Biology

country: USA

institute: Whitehead Instute - MIT

description:
Trizol isolation of total RNA from Undifferentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.399999999999999, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 21.806999999999999, Corner + 101, Corner -  101, Background 63.78, Noise 3.25, Raw Q 2.73
Keywords = adipocyte, subcutaneous, mesenteric, omental

molecule: total RNA

organism: Homo sapiens


title: Undifferentiated Omental Replicate 3B

description:
Trizol isolation of total RNA from Undifferentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.100000000000001, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 15.901, Corner + 57, Corner -  57, Background 46.80, Noise 1.94, Raw Q 2.02
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Female J110 - 1

city: Rennes

first: Daniel

last: Baron

state: Brittany

department: Station Commune de Recherches en Ichtyophysiologie

country: France

institute: Institut National de la Recherche Agronomique-INRA

description:
Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout
Animals and samplings:
Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.
Total RNA extraction and Reverse Transcription:
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.
Primers design:
Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.
Real-time RT-PCR
Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.

organism: Oncorhynchus mykiss

source_name: Female J110 - 1


title: Female J90 - 2

source_name: Female J90 - 2


title: Differentiated Subcutaneous Replicate 1B

description:
Trizol isolation of total RNA from Differentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.899999999999999, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 7.7290000000000001, Corner + 83, Corner -  83, Background 55.57, Noise 2.66, Raw Q 2.52
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Male J0 - 2

source_name: Male J0 - 2


title: Female J60 - 2

source_name: Female J60 - 2


title: Female J60 - 1

source_name: Female J60 - 1


title: Undifferentiated Omental Replicate 1B

description:
Trizol isolation of total RNA from Undifferentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.899999999999999, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 18.568000000000001, Corner + 51, Corner -  51, Background 50.36, Noise 1.88, Raw Q 2.1099999999999999
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 3B

description:
Trizol isolation of total RNA from Differentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.399999999999999, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 9.5920000000000005, Corner + 90, Corner -  90, Background 61.91, Noise 2.92, Raw Q 2.6600000000000001
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Mesenteric Replicate 3B

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.100000000000001, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 16.843, Corner + 130, Corner -  130, Background 73.03, Noise 3.78, Raw Q 2.98
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Female J7 - 1

source_name: Female J7 - 1


title: Female J12 - 1

source_name: Female J12 - 1


title: Female J0 - 2

source_name: Female J0 - 2


title: Female J27 - 1

source_name: Female J27 - 1


title: Undifferentiated Mesenteric Replicate 2B

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.399999999999999, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 13.352, Corner + 173, Corner -  173, Background 97.06, Noise 5.44, Raw Q 3.5100000000000002
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Female J7 - 2

source_name: Female J7 - 2


title: Undifferentiated Subcutaneous Replicate 3B

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.100000000000001, Fasting Glucose 102 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 16.206, Corner + 76, Corner -  76, Background 50.34, Noise 2.16, Raw Q 2.1899999999999999
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Subcutaneous Replicate 2B

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.399999999999999, Fasting Glucose 99 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 13.297000000000001, Corner + 75, Corner -  75, Background 61.84, Noise 2.63, Raw Q 2.4500000000000002
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Female J12 - 2

source_name: Female J12 - 2


title: Male J60 - 2

source_name: Male J60 - 2


title: Male J110 - 1

source_name: Male J110 - 1


title: Male J110 - 2

source_name: Male J110 - 2


title: Male J90 - 1

source_name: Male J90 - 1


title: Female J0 - 1

source_name: Female J0 - 1


title: Male J90 - 2

source_name: Male J90 - 2


title: Undifferentiated Mesenteric Replicate 1B

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.899999999999999, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 17.981000000000002, Corner + 59, Corner -  59, Background 53.87, Noise 2.20, Raw Q 2.3199999999999998
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Subcutaneous Replicate 1B

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.899999999999999, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 24.731000000000002, Corner + 50, Corner -  50, Background 46.55, Noise 1.56, Raw Q 1.9299999999999999
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Male J60 - 1

source_name: Male J60 - 1


title: Male J0 - 1

source_name: Male J0 - 1


title: Male J27 - 2

source_name: Male J27 - 2


title: Normal Peritoneal Macrophages/Neutrophils

status: Public on May 31 2005

city: Los Angeles

first: Adrian

middle: Friedrich

last: Gombart

state: CA

institute: Cedars-Sinai Medical Center

description:
Experimental Design
Prior studies of C/EBPe-deficient myeloid cells from both humans and mice, demonstrated an essential role for C/EBPe in the normal development and function of both neutrophils and macrophages. There are striking parallels between human neutrophil specific granule disease (SGD) due to mutations in the CEBPE gene and the murine C/EBPe-deficient condition.  This indicates that the C/EBPe-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease.  In this study, we sought to define further the role that C/EBPe plays in mediating host immune function by identifying possible target genes of C/EBPe in both neutrophils and macrophages.  This study was designed to elucidate the effects of the CEBPE gene knockout in murine myeloid cells.

Mice and sample preparation
The C/EBPE wild type (+/+) and –deficient (-/-) mice (129/SvEv x NIH Black Swiss) were generously provided by K.G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA).  They were maintained in pathogen-free facilities on normal chow.  To prepare RNA for array hybridization, four age-matched (6-8 weeks) mice of each genotype received an intraperitoneal (IP) injection of 2 ml of 4% sterile thioglycollate broth (Sigma Chemical Co., St Louis, MO).  At 24 h post injection, all mice were sacrificed and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice.  Total cell numbers were counted and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa stained cytospins (100-200 cells per sample) using a light microscope.   The preparations were composed of approximately 30% monocytes, 60% neutrophils and 7-10% lymphocytes in both the C/EBPe+/+ (wt) and C/EBPe-/- (ko) mice.  To reduce individual variation in subsequent experiments, the cells either from all wild type or all C/EBPe-null mice were pooled prior to probe synthesis (n=1 pooled RNA for wt and n=1 pooled RNA for ko)
Total RNA and protein was isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), Dnase I treated (Promega Corporation, Madison, WI) and purified using an RNeasy spin-column (Qiagen, Valencia, CA).  The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel.

Hybridization and analysis of the Affymetrix GeneChip Murine 11K Set.  The murine 11K set consists of 2 probe arrays (subarray A and B) that allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene (8/96 and Build 4.0) and TIGR (Build 1.0 beta) databases (Affymetrix Inc, Santa Clara, CA).  Biotinylated cRNAs were prepared from the pooled RNA samples and fragmented following the manufacturer’s protocol (Affymetrix Inc.).  The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.).  The probed arrays (n=1) were scanned with a Hewlett Packard Gene Array scanner.  The scanned images were analyzed to generate the quantitations based on the images using GeneChip 3.1 software (Affymetrix Inc.).  The default settings of the software were used for the analysis.  Below are the algorithm parameters for each array.
Algorithm Parameters:	GEO WT_11KSubB
BF=	SDT=237.9(4.00Q)	SRT=1.50	ABS={{3.0,4.0},{0.33,0.43},{0.90,1.30}}	RawQ=17.40
QMult=2.80	RL=10.0	HZ=4	VZ=4	BG=2
CT=237.9	PCT=80	DD={{3.0,4.0},{0.33,0.43},{0.20,0.30},{0.90,1.30}}	AvgNCT=200	FCMin=1.00
FCMax=1.00	STP=3.0	TGT=2500	NF=1.000000	SF=3.418797
SFGene=All

Experiment:	WT-subB
Corner+		Avg:1491		Count:32
Corner-		Avg:45651		Count:32
Central-		Avg:45651		Count:9
Edge+		Avg:1151		Count:8
Edge-		Avg:45651		Count:8
Background	Avg: 504	Stdv: 16.7	Min: 475	Max: 528
Pixels	Avg: 36	Stdv: 1.1	Min: 25	Max: 36
Keywords = CEBPE peritoneal macrophage neutrophil knockout

organism: Mus musculus

source_name: Total RNA from peritoneal cells harvested by lavage 24 h post thioglycollate injection


title: C/EBP epsilon-deficient Peritoneal Macrophages/Neutrophils

description:
Experimental Design
Prior studies of C/EBPe-deficient myeloid cells from both humans and mice, demonstrated an essential role for C/EBPe in the normal development and function of both neutrophils and macrophages. There are striking parallels between human neutrophil specific granule disease (SGD) due to mutations in the CEBPE gene and the murine C/EBPe-deficient condition.  This indicates that the C/EBPe-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease.  In this study, we sought to define further the role that C/EBPe plays in mediating host immune function by identifying possible target genes of C/EBPe in both neutrophils and macrophages.  This study was designed to elucidate the effects of the CEBPE gene knockout in murine myeloid cells.

Mice and sample preparation
The C/EBPE wild type (+/+) and –deficient (-/-) mice (129/SvEv x NIH Black Swiss) were generously provided by K.G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA).  They were maintained in pathogen-free facilities on normal chow.  To prepare RNA for array hybridization, four age-matched (6-8 weeks) mice of each genotype received an intraperitoneal (IP) injection of 2 ml of 4% sterile thioglycollate broth (Sigma Chemical Co., St Louis, MO).  At 24 h post injection, all mice were sacrificed and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice.  Total cell numbers were counted and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa stained cytospins (100-200 cells per sample) using a light microscope.   The preparations were composed of approximately 30% monocytes, 60% neutrophils and 7-10% lymphocytes in both the C/EBPe+/+ (wt) and C/EBPe-/- (ko) mice.  To reduce individual variation in subsequent experiments, the cells either from all wild type or all C/EBPe-null mice were pooled prior to probe synthesis (n=1 pooled RNA for wt and n=1 pooled RNA for ko)
Total RNA and protein was isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), Dnase I treated (Promega Corporation, Madison, WI) and purified using an RNeasy spin-column (Qiagen, Valencia, CA).  The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel.

Hybridization and analysis of the Affymetrix GeneChip Murine 11K Set.  The murine 11K set consists of 2 probe arrays (subarray A and B) that allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene (8/96 and Build 4.0) and TIGR (Build 1.0 beta) databases (Affymetrix Inc, Santa Clara, CA).  Biotinylated cRNAs were prepared from the pooled RNA samples and fragmented following the manufacturer’s protocol (Affymetrix Inc.).  The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.).  The probed arrays (n=1) were scanned with a Hewlett Packard Gene Array scanner.  The scanned images were analyzed to generate the quantitations based on the images using GeneChip 3.1 software (Affymetrix Inc.).  The default settings of the software were used for the analysis.  Below are the algorithm parameters for each array.
Algorithm Parameters:	GEO KO_11KSubA
BF=	SDT=311.2(4.00Q)	SRT=1.50	ABS={{3.0,4.0},{0.33,0.43},{0.90,1.30}}	RawQ=20.33
QMult=2.80	RL=10.0	HZ=4	VZ=4	BG=2
CT=311.2	PCT=80	DD={{3.0,4.0},{0.33,0.43},{0.20,0.30},{0.90,1.30}}	AvgNCT=200	FCMin=1.00
FCMax=1.00	STP=3.0	TGT=2500	NF=1.000000	SF=3.827028
SFGene=All

Experiment:	KO-subA
Corner+		Avg:1501		Count:32
Corner-		Avg:45559		Count:32
Central-		Avg:45559		Count:9
Edge+		Avg:1048		Count:8
Edge-		Avg:45559		Count:8
Background	Avg: 579	Stdv: 20.3	Min: 553	Max: 615
Pixels	Avg: 36	Stdv: 1.1	Min: 25	Max: 36
Keywords = CEBPE peritoneal macrophage neutrophil knockout

source_name: Total RNA from C/EBP epsilon-deficient peritoneal cells harvested by lavage 24 h post thioglycollate injection


description:
Experimental Design
Prior studies of C/EBPe-deficient myeloid cells from both humans and mice, demonstrated an essential role for C/EBPe in the normal development and function of both neutrophils and macrophages. There are striking parallels between human neutrophil specific granule disease (SGD) due to mutations in the CEBPE gene and the murine C/EBPe-deficient condition.  This indicates that the C/EBPe-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease.  In this study, we sought to define further the role that C/EBPe plays in mediating host immune function by identifying possible target genes of C/EBPe in both neutrophils and macrophages.  This study was designed to elucidate the effects of the CEBPE gene knockout in murine myeloid cells.

Mice and sample preparation
The C/EBPE wild type (+/+) and –deficient (-/-) mice (129/SvEv x NIH Black Swiss) were generously provided by K.G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA).  They were maintained in pathogen-free facilities on normal chow.  To prepare RNA for array hybridization, four age-matched (6-8 weeks) mice of each genotype received an intraperitoneal (IP) injection of 2 ml of 4% sterile thioglycollate broth (Sigma Chemical Co., St Louis, MO).  At 24 h post injection, all mice were sacrificed and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice.  Total cell numbers were counted and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa stained cytospins (100-200 cells per sample) using a light microscope.   The preparations were composed of approximately 30% monocytes, 60% neutrophils and 7-10% lymphocytes in both the C/EBPe+/+ (wt) and C/EBPe-/- (ko) mice.  To reduce individual variation in subsequent experiments, the cells either from all wild type or all C/EBPe-null mice were pooled prior to probe synthesis (n=1 pooled RNA for wt and n=1 pooled RNA for ko)
Total RNA and protein was isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), Dnase I treated (Promega Corporation, Madison, WI) and purified using an RNeasy spin-column (Qiagen, Valencia, CA).  The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel.

Hybridization and analysis of the Affymetrix GeneChip Murine 11K Set.  The murine 11K set consists of 2 probe arrays (subarray A and B) that allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene (8/96 and Build 4.0) and TIGR (Build 1.0 beta) databases (Affymetrix Inc, Santa Clara, CA).  Biotinylated cRNAs were prepared from the pooled RNA samples and fragmented following the manufacturer’s protocol (Affymetrix Inc.).  The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.).  The probed arrays (n=1) were scanned with a Hewlett Packard Gene Array scanner.  The scanned images were analyzed to generate the quantitations based on the images using GeneChip 3.1 software (Affymetrix Inc.).  The default settings of the software were used for the analysis.  Below are the algorithm parameters for each array.
Algorithm Parameters:	GEO KO_11KSubB
BF=	SDT=235.4(4.00Q)	SRT=1.50	ABS={{3.0,4.0},{0.33,0.43},{0.90,1.30}}	RawQ=20.82
QMult=2.80	RL=10.0	HZ=4	VZ=4	BG=2
CT=235.4	PCT=80	DD={{3.0,4.0},{0.33,0.43},{0.20,0.30},{0.90,1.30}}	AvgNCT=200	FCMin=1.00
FCMax=1.00	STP=3.0	TGT=2500	NF=1.000000	SF=2.826455
SFGene=All

Experiment:	KO-subB
Corner+		Avg:1437		Count:32
Corner-		Avg:45499		Count:32
Central-		Avg:45499		Count:9
Edge+		Avg:1329		Count:8
Edge-		Avg:45499		Count:8
Background	Avg: 634	Stdv: 41.7	Min: 548	Max: 694
Pixels	Avg: 36	Stdv: 1.2	Min: 25	Max: 36
Keywords = CEBPE peritoneal macrophage neutrophil knockout


title: HR vs LR (M5-84)

status: Public on May 26 2005

city: Baton Rouge

first: Leslie

middle: DeBardeleben

last: McLaughlin

state: LA

address: 6400 Perkins Road

department: Department of Experimental Obesity

institute: Pennington Biomedical Research Center

description:
Source1 (Channel 1; Cy5)= Low Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 LR (ran <= 2 km/night).

Source2 (Channel 2; Cy3)= High Runners (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >=5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the high runners (n=6) HR and each of the low runners (n=6) LR were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. NOTE: One slide from this series broke during processing, so there were only 5 (2 forward-labeled, 3 reverse-labeled) for this series. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M5-84

source_name: pooled hippocampal RNA from LR

source_name: pooled hippocampal RNA from HR


title: sed vs MR (M6-62)

description:
Source1 (Channel 1; Cy5)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Source2 (Channel 2; Cy3)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-62

source_name: pooled hippocampal RNA from MR

source_name: pooled hippocampal RNA from sedentary controls


title: Female J110 - 2

source_name: Female J110 - 2


title: sed vs HR (M6-44)

description:
Source2 (Channel 2; Cy3)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source1 (Channel 1; Cy5)= High Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >= 5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the top (n=6) HR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Lot batch = PBRC M6-44


title: LR vs HR (M5-87)

description:
Source2 (Channel 2; Cy3)= Low Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 LR (ran <= 2 km/night).

Source1 (Channel 1; Cy5)= High Runners (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >=5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the high runners (n=6) HR and each of the low runners (n=6) LR were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. NOTE: One slide from this series broke during processing, so there were only 5 (2 forward-labeled, 3 reverse-labeled) for this series. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M5-87


title: LR vs HR (M5-86)

description:
Source2 (Channel 2; Cy3)= Low Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 LR (ran <= 2 km/night).

Source1 (Channel 1; Cy5)= High Runners (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >=5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the high runners (n=6) HR and each of the low runners (n=6) LR were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. NOTE: One slide from this series broke during processing, so there were only 5 (2 forward-labeled, 3 reverse-labeled) for this series. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M5-86


title: MR vs sed (M6-61)

description:
Source1 (Channel 1; Cy3)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Source2 (Channel 2; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-61


title: HR vs sed (M6-47)

description:
Source1(Channel 1; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source2 (Channel 2; Cy3)= High Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >= 5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the top (n=6) HR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-47


title: sed vs MR (M6-59)

description:
Source1 (Channel 1; Cy5)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Source2 (Channel 2; Cy3)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-59


title: sed vs HR (M6-46)


title: MR vs sed (M6-60)

description:
Source1 (Channel 1; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source2 (Channel 2; Cy3)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-60


title: LR vs HR (M5-88)

description:
Source2 (Channel 2; Cy3)= Low Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 LR (ran <= 2 km/night).

Source1 (Channel 1; Cy5)= High Runners (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >=5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the high runners (n=6) HR and each of the low runners (n=6) LR were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. NOTE: One slide from this series broke during processing, so there were only 5 (2 forward-labeled, 3 reverse-labeled) for this series. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M5-88


title: HR vs LR (M5-30)

description:
Source1 (Channel 1; Cy5)= Low Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 LR (ran <= 2 km/night).

Source2 (Channel 2; Cy3)= High Runners (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >=5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the high runners (n=6) HR and each of the low runners (n=6) LR were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. NOTE: One slide from this series broke during processing, so there were only 5 (2 forward-labeled, 3 reverse-labeled) for this series. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M5-30


title: Female J27 - 2

description:
Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout
Animals and samplings:
Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.
Total RNA extraction and Reverse Transcription:
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.
Primers design:
Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.
Real-time RT-PCR
Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.

source_name: Female J27 - 2


title: HR vs sed (M6-43)

description:
Source1 (Channel 1; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source2 (Channel 2; Cy3)= High Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >= 5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit.  Equal amounts (6-mg) of total RNA from each of the top (n=6) HR and sedentary controls (n=6) were combined to generate 2 separate pools.  Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides.  Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison.  The protocol for labeling and hybridization was basically as follows.   The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-43