title: Undifferentiated Omental Replicate 3

description:
Trizol isolation of total RNA from Undifferentiated Human Omental Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, 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 3.658, Corner + 64, Corner -  64, Background 61.21, Noise 2.77, Raw Q 2.52
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Omental Replicate 1

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, 31 YO, BMI 47.9, 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 3.493, Corner + 70, Corner -  70, Background 64.37, Noise 2.77, Raw Q 2.53
Keywords = adipocyte, subcutaneous, mesenteric, omental

molecule: total RNA

organism: Homo sapiens


title: F JH 4 h 150837

status: Public on Jun 16 2005

city: Reno

first: Christopher

middle: Ian

last: Keeling

state: NV

address: 1664 North Virginia Street

department: Department of Biochemistry/Mail Stop 330

institute: University of Nevada, Reno

description:
Tissue: Logging slash of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) infested with Ips pini were obtained from Ward Creek CA (39*08'41" N, 120*12'01" W, 2048 m) and placed in a greenhouse to allow emergence of adult beetles (Browne, J. Econ. Entomol. 65, 1499-1501, 1972). Upon emergence, beetles were typed and sexed and kept at 4°C on moist paper towels. Beetles were immobilized by chilling and then treated topically on the ventral surface of the abdomen with either 10 µg (±) juvenile hormone III (Sigma) in 0.5 µL acetone or acetone (control). They were then incubated in the dark at room temperature in groups of 10 in 60 mL plastic containers (Dixie) for specified times.  At the end of the incubation, beetles were immersed in water and the anterior midguts excised under a stereo microscope. Midguts were gently purged of their contents and then immediately frozen in liquid nitrogen and stored at -80°C. Each treatment/control time point pair was replicated four times and each replicate contained tissue from 60 midguts (30 each for treatment and control). Treatment and control beetles were cohorts for a replicate pair and were incubated simultaneously. Beetles in different replicate pairs and time points were not always cohorts.
Hybridizations: Hybridizations were done in quadruplicate, each with tissue from different groups of beetles. Total RNA was extracted using QIAshredder and RNeasy Mini kits (Qiagen) and quantified spectrophotometrically. Total RNA (approx. 40 µg) was reverse transcribed with oligo(dT)15 (Promega), Superscript III RT- (Invitrogen), and dNTPs (Promega) containing aminoallyl-dUTP (Sigma, aminoallyl-dUTP to dTTP ratio of 2 to 3). After alkaline hydrolysis of the RNA, clean up with a Microcon-30 (Millipore), and lyophilization, the aminoallyl-dUTP cDNA was reacted with Cy3 or Cy5 monofunctional reactive dyes (Amersham). Cy3 and Cy5 dyes were swapped equally between treatment and control samples among replicates (see source info for dye used for each target). After 60 min, unreacted dyes were removed from the labeled cDNA by a QIAquick spin column (Qiagen). The fluorescently labeled cDNAs from treatment and control samples were combined together with 20 µg herring sperm DNA (Promega) and 20 µg polydA (Amersham), lyophilized, and then resuspended in 20 µL hybridization buffer (50% formamide, 5x SSC, and 0.1% SDS). The hybridization mixture was denatured at 95°C for 5 min, centrifuged at 16,000 g for 5 min, and then immediately applied to the microarray preheated to 42°C. Prior to hybridization, microarrays were washed 5 min in 0.1% SDS, 5 min in 2x SSC, denatured 3 min in boiling water, placed into ice-cold ethanol, dried by centrifugation, incubated for 60 min at 42°C in pre-hybridization buffer (5x SSC, 0.1% SDS, and 1% BSA), washed 5 min in water, 5 min in 2-propanol, and then dried by centrifugation. The hybridization mixture was covered with a HybriSlip (22 x 40 mm, Sigma) and then the microarray was immediately placed into a preheated, humidified hybridization chamber and placed in a 42°C oven for 16-20 h. Microarrays were removed from the hybridization chamber and immediately washed with agitation at room temperature for 5 min each in 2x SSC with 0.03% SDS, 1x SSC, and 0.2x SSC, and then dried by centrifugation.
Analysis: Arrays were scanned at 5 µm resolution with a Packard Bioscience ScanArray 4000 array scanner and analyzed with ScanArray and QuantArray software (versions 3.1 and 3.0 respectively). Median signal (adaptive spot algorithm) and background intensities were obtained for both dyes for each element. Data were background subtracted, globally and locally (across microarray surface, span=0.5, trim=0.1) normalized, log transformed (base 2), converted to mean log (intensities) and log (ratios), and then locally normalized across element signal intensity (span=0.7, trim=0.1) using online SNOMAD tools (Colantuoni et al., Bioinformatics 18, 1540-1, 2002). "Empty" and "Spotting Soln" elements were excluded from analysis by SNOMAD.  Data for these two element types were arbitrarily filled with zeros to upload into NCBI GEO.  To account for outliers, the elements in triplicate on the 4 replicated arrays were treated as unique measurements and a Q-test (96% confidence, Dean and Dixon, Anal. Chem. 23, 636-8, 1951) was performed on the 12 measurements. If a measurement tested as an outlier, its VALUE was replaced with the average VALUE of the two other replicate elements on the same array.  At most, only 1 of the 12 measurements was excluded.
Keywords = North American pine engraver beetle, anterior midgut, juvenile hormone, pheromone biosynthesis, Coleoptera, Scolytidae

organism: Ips pini

source_name: Anterior midguts from acetone treated females, 4 hours (Cy3)

source_name: Anterior midguts from juvenile hormone III treated females, 4 hours (Cy5)


title: IRS-4 KO (jos0931)

city: Boston

first: Atul

last: Butte

address: 300 Longwood Avenue

institute: Children's Hospital, Boston

description:
Brown preadipocyte cell lines derived from newborn IRS-4 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 931

organism: Mus musculus

source_name: Mouse brown preadipocyte cell line


title: Undifferentiated Mesenteric Replicate 2

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, 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 4.407, Corner + 55, Corner -  55, Background 61.28, Noise 2.45, Raw Q 2.45
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: IRS-3 KO (jos0953)

description:
Brown preadipocyte cell lines derived from newborn IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 953


title: IRS-4 KO (jos0954)

description:
Brown preadipocyte cell lines derived from newborn IRS-4 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 954


title: Undifferentiated Mesenteric Replicate 3

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, 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 4.171, Corner + 118, Corner -  118, Background 91.90, Noise 5.43, Raw Q 3.53
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Subcutaneous Replicate 2

description:
Trizol isolation of total RNA from Differentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, 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 2.868, Corner + 75, Corner -  75, Background 55.74, Noise 2.74, Raw Q 2.40
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: IRS-2 KO (jos1527)

description:
Brown preadipocyte cell lines derived from newborn IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1527


title: IRS-3 KO (jos1528)

description:
Brown preadipocyte cell lines derived from newborn IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1528


title: Undifferentiated Mesenteric Replicate 1

description:
Trizol isolation of total RNA from Undifferentiated Human Mesenteric Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.9, 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 4.952, Corner + 79, Corner -  79, Background 74.85, Noise 3.81, Raw Q 3.41
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Undifferentiated Subcutaneous Replicate 1

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.9, 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 7.370, Corner + 48, Corner -  48, Background 56.50, Noise 2.09, Raw Q 2.24
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Subcutaneous Replicate 3

description:
Trizol isolation of total RNA from Differentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, 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 3.476, Corner + 80, Corner -  80, Background 61.91, Noise 2.97, Raw Q 2.51
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: IRS-2 KO (jos0952)

description:
Brown preadipocyte cell lines derived from newborn IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 952


title: Undifferentiated Subcutaneous Replicate 3

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 38 YO, BMI 51.1, 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 4.146, Corner + 62, Corner -  62, Background 61.22, Noise 2.47, Raw Q 2.41
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: IRS-3 KO (jos0930)

description:
Brown preadipocyte cell lines derived from newborn IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 930


title: IRS-2 KO (jos0929)

description:
Brown preadipocyte cell lines derived from newborn IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 929


title: Undifferentiated Subcutaneous Replicate 2

description:
Trizol isolation of total RNA from Undifferentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 29 YO, BMI 44.4, 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 4.783, Corner + 123, Corner -  123, Background 83.97, Noise 4.53, Raw Q 3.22
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: IRS-4 KO (jos0700)

description:
Brown preadipocyte cell lines derived from newborn IRS-4 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 700


title: Normal Lung 2

first: Gavin

last: Gordon

institute: Brigham and Women's Hospital/Harvard Medical School

description: Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Boston’s Brigham and Women’s Hospital (BWH) between October 1998 and August 2000.  All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed.  All normal specimens were obtained from patients who were never diagnosed with MPM.  Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH.  The JMN1B MPM cell line19,20 has been described previously.  The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection.  Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM.  All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used).  The microscopic slides from the patients’ resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases.  Linked clinical and pathological data were obtained for all patients who contributed tumor specimens.  Specimens and data were rendered anonymous to protect patient confidentiality.  Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH.

source_name: Normal Lung 2


title: IRS-3 KO (jos0699)

description:
Brown preadipocyte cell lines derived from newborn IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 699


title: IRS-4 KO (jos1529)

description:
Brown preadipocyte cell lines derived from newborn IRS-4 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1529


title: IRS-2 KO (jos0698)

description:
Brown preadipocyte cell lines derived from newborn IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 698


title: IRS-1 KO (jos0669)

description:
Brown preadipocyte cell lines derived from newborn IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 669


title: Normal Pleura 5

source_name: Normal Pleura 5


title: IRS-1 KO (jos1534)

description:
Brown preadipocyte cell lines derived from newborn IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1534


title: IRS-1 KO (jos0649)

description:
Brown preadipocyte cell lines derived from newborn IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 649


title: Wildtype 3 (jos1525)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1525


title: Wildtype 3 (jos0534)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 534


title: Wildtype 2 (jos0927)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 927


title: Wildtype 2 (jos1524)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse


title: Wildtype 2 (jos0533)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 533


title: Wildtype 1 (jos1523)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 1523


title: Wildtype 2 (jos0950)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-2 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 950


title: Wildtype 1 (jos0668)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation.  Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described.  15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA).  Intensity values were quantitated by MAS 5.0 software (Affymetrix).  All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 668


title: Male J27 - 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: Male J27 - 1


title: Male J12 - 2

source_name: Male J12 - 2


title: Male J7 - 2

source_name: Male J7 - 2


title: Male J12 - 1

source_name: Male J12 - 1


title: IRS-1 KO (jos0678)

description:
Brown preadipocyte cell lines derived from newborn IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 678


title: Wildtype 1 (jos0677)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation.  Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described.  15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA).  Intensity values were quantitated by MAS 5.0 software (Affymetrix).  All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 677


title: Wildtype 3 (jos0951)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-3 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 951


title: Differentiated Omental Replicate 3B

description:
Trizol isolation of total RNA from Differentiated 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 10.089, Corner + 93, Corner -  93, Background 61.86, Noise 3.25, Raw Q 2.73
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Omental Replicate 1B

description:
Trizol isolation of total RNA from Differentiated 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 8.8420000000000005, Corner + 108, Corner -  108, Background 67.91, Noise 3.46, Raw Q 2.8300000000000001
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Male J7 - 1

source_name: Male J7 - 1


title: Differentiated Omental Replicate 2B

description:
Trizol isolation of total RNA from Differentiated 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 9.4849999999999994, Corner + 96, Corner -  96, Background 59.49, Noise 3.16, Raw Q 2.7200000000000002
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 2B

description:
Trizol isolation of total RNA from Differentiated 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 9.0380000000000003, Corner + 88, Corner -  88, Background 58.45, Noise 2.98, Raw Q 2.7400000000000002
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Subcutaneous Replicate 3B

description:
Trizol isolation of total RNA from Differentiated 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 12.959, Corner + 89, Corner -  89, Background 60.41, Noise 2.81, Raw Q 2.6200000000000001
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: Differentiated Mesenteric Replicate 1B

description:
Trizol isolation of total RNA from Differentiated 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 6.8380000000000001, Corner + 87, Corner -  87, Background 52.12, Noise 2.54, Raw Q 2.3700000000000001
Keywords = adipocyte, subcutaneous, mesenteric, omental