title: AJ_HF_s70_Week7

description:
AJ mouse, total liver RNA, high-fat diet, Week7
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week7


title: AJ_HF_s69_Week7

status: Public on Jul 31 2004

type: RNA

city: Cambridge

first: Jatin

last: Misra

state: MA

country: USA

institute: MIT

molecule: total RNA

organism: Mus musculus

source_name: control, Cy3 dye, mixture of total RNA from liver, skeletal muscle, brain, heart, lung, testes, hypothalamus, brown adipose tissue, white adipose tissue, and kidney of C57 mice'


title: AJ_HF_s68_Week7


title: AJ_LF_s21_Week3

description:
AJ mouse, total liver RNA, low-fat diet, Week3
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, low-fat diet, Week3


title: AJ_HF_s63_Week7


title: AJ_HF_s62_Week7


title: AJ_HF_s60_Week6

description:
AJ mouse, total liver RNA, high-fat diet, Week6
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week6


title: AJ_HF_s59_Week6


title: AJ_HF_s56_Week6


title: AJ_HF_s55_Week6


title: AJ_HF_s65_Week7


title: AJ_HF_s57_Week6


title: AJ_HF_s64_Week7


title: AJ_HF_s66_Week7


title: AJ_HF_s67_Week7


title: AJ_HF_s58_Week6


title: AJ_HF_s61_Week7


title: AJ_HF_s54_Week6


title: AJ_HF_s52_Week6


title: AJ_HF_s45_Week5

description:
AJ mouse, total liver RNA, high-fat diet, Week5
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week5


title: AJ_HF_s53_Week6


title: AJ_HF_s46_Week5


title: AJ_HF_s49_Week5


title: AJ_HF_s48_Week5


title: AJ_HF_s44_Week5


title: AJ_HF_s51_Week6


title: AJ_HF_s50_Week5


title: AJ_HF_s43_Week5


title: AJ_HF_s42_Week5


title: AJ_HF_s47_Week5


title: AJ_HF_s41_Week5


title: AJ_HF_s26_Week3

description:
AJ mouse, total liver RNA, high-fat diet, Week3
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week3


title: AJ_HF_s30_Week3


title: AJ_HF_s39_Week4

description:
AJ mouse, total liver RNA, high-fat diet, Week4
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week4


title: AJ_HF_s32_Week4


title: AJ_HF_s40_Week4


title: AJ_HF_s38_Week4


title: AJ_HF_s34_Week4


title: AJ_HF_s33_Week4


title: AJ_HF_s37_Week4


title: AJ_HF_s31_Week4


title: AJ_HF_s28_Week3


title: AJ_HF_s36_Week4


title: AJ_HF_s27_Week3


title: AJ_HF_s24_Week3


title: AJ_HF_s35_Week4


title: AJ_HF_s16_Week1

description:
AJ mouse, total liver RNA, high-fat diet, Week1
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week1


title: AJ_HF_s20_Week1


title: AJ_HF_s25_Week3


title: AJ_HF_s21_Week3


title: AJ_HF_s19_Week1


title: AJ_HF_s17_Week1


title: AJ_HF_s23_Week3


title: AJ_HF_s29_Week3


title: AJ_HF_s22_Week3


title: AJ_HF_s15_Week1


title: AJ_HF_s14_Week1


title: AJ_HF_s18_Week1


title: AJ_HF_s9_Week0

description:
AJ mouse, total liver RNA, high-fat diet, Week0
The liver samples were homogenized (1ml/50mg tissue) in RNA STAT-60 (Tel-Test, Friendswood, TX) with a Tissue-TearorTM  (Biospec Products, Bartlesville, OK).  RNA was isolated according to the manufacturer recommended protocol.  Briefly, once the tissue was homogenized, RNA was extracted through chloroform addition, which separated the STAT-60 reagent into a heavy phenol chloroform phase and a light colorless upper aqueous phase, which contained the RNA.  The aqueous phase was removed and mixed with isopropanol in order to precipitate RNA.  The precipitated RNA was washed with 75% ethanol, and then dried for 5-10 minutes.  The RNA pellet was then dissolved in nuclease-free water (Ambion).  Once the pellet was completely dissolved, the RNA was cleaned using the Qiagen RNEasy Mini Kit (Catalog # 74104, Qiagen, Maryland, USA) in order to remove any traces of ethanol that may interfere with the reverse transcription reaction.
For the control, RNA from the following C57 mouse tissues were pooled in the proportions indicated in parentheses: white adipose tissue (5%), skeletal muscle (12%), brain (4%), heart(1%), lung (6%), testes (2%), hypothalamus (6%), brown adipose tissue (13%), kidney (26%), and liver (26%).  This was done in order to maximize the number of genes represented in the control, and thereby enable the quantification of as many spots as possible on the array.
The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60 ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42 ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples were then combined with their controls. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65 ºC. Following concentration, the sample was resuspended in 40 uL of prewarmed hybridization buffer (Clontech Laboratories), and hybridized to duplicate oligonucleotide microarrays, and 20 ul was dispensed on each array. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.).
The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene).

source_name: experiment, Cy5 dye, liver total RNA, high-fat diet, Week0


title: AJ_HF_s11_Week1


title: AJ_HF_s13_Week1


title: AJ_HF_s5_Week0


title: vehicle #2

status: Public on Jul 30 2004

city: Madison

first: Thor

middle: D

last: Stein

state: WI

institute: University of Wisconsin-Madison

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 13 days and treated with phosphate buffered saline (vehicle) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1 mM glutamine.  The sample consists of 8 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha

source_name: hippocampal slice cultures


title: sAPPalpha #3

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 11 days and treated with 1 nM sAPPalpha (sAPPalpha) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1 mM glutamine.  The sample consists of 11 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha


title: AJ_HF_s10_Week0


title: AJ_HF_s2_Week0


title: AJ_HF_s4_Week0


title: vehicle #3

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 11 days and treated with phosphate buffered saline (vehicle) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1 mM glutamine.  The sample consists of 10 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha


title: sAPPalpha #1

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 15 days and treated with 1 nM sAPPalpha (sAPPalpha) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1 mM glutamine.  The sample consists of 12 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha


title: AJ_HF_s3_Week0


title: AJ_HF_s6_Week0


title: sAPPalpha #2

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 13 days and treated with 1 nM sAPPalpha (sAPPalpha) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1 mM glutamine.  The sample consists of 9 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha


title: vehicle #1

description:
Organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL) were maintained in culture for 15 days and treated with phosphate buffered saline (vehicle) for an additional 24 h.  Cultures were maintained on filter inserts in Neurobasal media with B-27 supplement (Invitrogen) and 1mM glutamine.  The sample consists of 12 slices from 4 mice.  Data were analyzed with MAS 5.0 and scaled to 2500.
Keywords = Alzheimer’s disease
Keywords = neuroprotection
Keywords = sAPPalpha


title: slide44 24hours

status: Public on Jul 28 2004

city: San Francisco

first: Michael

middle: E.

last: Salazar

state: CA

address: 1550 Fourth Street, RM 545

institute: University of California, San Francisco

description:
For microarray analysis, total RNA was purified from quiescent C2C12 myoblasts and myoblasts that were stimulated with Ang II for 12 h or 24 h using the RNeasy Mini Kit (Qiagen)."
The quality of RNA was analyzed using the RNA 6000 NanoKit on an Agilent BioAnalyzer 2100 system (Agilent Technologies); as expected, discreet 28 S rRNA and 18 S rRNA peaks were observed."
RNA samples which had an OD260/ OD280 ratio of 1.8-2.1 were used.
The library used was the Qiagen/Operon v2.0 mouse oligo library, which is composed of oligonucleotides (70mers) representing 16,443 unique mouse genes."
Each library of oligonucleotides also included sets of positive and negative controls that were used for quality control purposes.
Targets to be hybridized to the arrays were prepared by synthesis of single stranded cDNA from total RNA using an oligo(dT) primer in the presence of amino-allyl modified dUTP.
After synthesis, RNA was removed by hydrolysis in alkali followed by neutralization and ethanol precipitation."
Either monofunctional NHS-ester Cy3 or Cy5 fluors were chemically coupled to the free amine groups incorporated into the cDNA.
After coupling, the uncoupled fluors were quenched by the addition of hydroxylamine and then removed by purification using a Qiagen QIAquick cleanup kit."
Efficiency of cDNA synthesis, and Cy3 and Cy5 labeling was monitored by measurement of the absorbance at 260, 550 and 649 nm, respectively."
Arrays were hybridized using a Genomic Solutions GeneTAC Hybridization Station according to the manufacturer’s instructions.
lides were scanned for the presence of Cy3 and Cy5 signal using an Axon GenePix4000 scanner and the images quantified using GenePix Pro 3.0 software.
Normalization of arrays was performed using the Locally-Weighted Scatter plot Smooth (LOWESS) normalization algorithm in the Bioconductor package

source_name: Control

source_name: Angiotensin II


title: AJ_HF_s8_Week0


title: AJ_HF_s1_Week0


title: slide42 12 vs 0 hour-1


title: slide39 0hour


title: slide34 24 vs 0 hour-2


title: slide50 0 vs 0 hour-1


title: slide33 12 vs 0 hour-2

description:
For microarray analysis, total RNA was purified from quiescent C2C12 myoblasts and myoblasts that were stimulated with Ang II for 12 h or 24 h using the RNeasy Mini Kit (Qiagen).
The quality of RNA was analyzed using the RNA 6000 NanoKit on an Agilent BioAnalyzer 2100 system (Agilent Technologies); as expected, discreet 28 S rRNA and 18 S rRNA peaks were observed.
RNA samples which had an OD260/ OD280 ratio of 1.8-2.1 were used.
The library used was the Qiagen/Operon v2.0 mouse oligo library, which is composed of oligonucleotides (70mers) representing 16,443 unique mouse genes.
Each library of oligonucleotides also included sets of positive and negative controls that were used for quality control purposes.
Targets to be hybridized to the arrays were prepared by synthesis of single stranded cDNA from total RNA using an oligo(dT) primer in the presence of amino-allyl modified dUTP.
After synthesis, RNA was removed by hydrolysis in alkali followed by neutralization and ethanol precipitation.
Either monofunctional NHS-ester Cy3 or Cy5 fluors were chemically coupled to the free amine groups incorporated into the cDNA.
After coupling, the uncoupled fluors were quenched by the addition of hydroxylamine and then removed by purification using a Qiagen QIAquick cleanup kit.
Efficiency of cDNA synthesis, and Cy3 and Cy5 labeling was monitored by measurement of the absorbance at 260, 550 and 649 nm, respectively.
Arrays were hybridized using a Genomic Solutions GeneTAC Hybridization Station according to the manufacturer’s instructions.
lides were scanned for the presence of Cy3 and Cy5 signal using an Axon GenePix4000 scanner and the images quantified using GenePix Pro 3.0 software.
Normalization of arrays was performed using the Locally-Weighted Scatter plot Smooth (LOWESS) normalization algorithm in the Bioconductor package


title: slide43 12hours


title: HUVEC 2,5hr IL1

status: Public on Jul 20 2004

city: Vienna

first: Herbert

last: Mayer

address: Brunnerstr 59

country: Austria

institute: Institute of Vascular Biology and Thrombosis Research

description: Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4)  at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500. The given sample record represents the 2,5 hr time point of IL-1 stimulation.

organism: Homo sapiens

source_name: Total RNA from IL-1 stimulated HUVEC (2,5hr)


title: HUVEC 1hr IL1

description: Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4)  at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500. The given sample record represents the 1 hr time point of IL-1 stimulation.

source_name: Total RNA from IL-1 stimulated HUVEC (1hr)


title: KC_Hyb01_F085_7K8C_t72

status: Public on Jul 03 2004

city: Kiel

first: Timothy

middle: TeHua

last: Lu

country: Germany

institute: Christian-Albrechts-University Kiel

description:
Target labeling and hybridization were conducted according manufacturers protocols. http://www.clontech.com/atlas/index.shtml

Spot quantitation:
Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate). For this dataset, the weight of each pixel was set to one.

organism: Rattus norvegicus

source_name: Cerebellum tissue : normal, healthy rat


title: AJ_HF_s12_Week1


title: HC_Hyb15_F251_38H8C_t70

source_name: Cerebellum tissue : Harmaline treated rat


title: HUVEC 6hr IL1

description: Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4)  at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500. The given sample record represents the 6 hr time point of IL-1 stimulation.

source_name: Total RNA from IL-1 stimulated HUVEC (6hr)


title: slide60 24 vs 0 hour-1


title: HC_Hyb02_F044_5H8C_t24


title: Hepatic reductase null N8

status: Public on Jul 11 2004

city: Dundee

first: Wang

middle: Jun

last: Xiu

address: Ninewells Hospital

department: Biomedical Research Centre

country: United Kingdom

institute: Ninewells Hospital Medical School

description:
Mice were sacrificed by a rising concentration of CO2 and livers were immediately removed for preparation of RNA. Total RNA was isolated from mouse liver with a phenol-guanidine isothiocyanate reagent, TRIzol (Invitrogen) and further purified with RNeasy Mini Kit (Qiagen Ltd) in accordance with the manufacturer’s instructions. RNA was pooled from three animals of each genotype for each experiment, and the experiments were carried out twice. Results from the two experiments were exactly the same. The A260/280 ratio of total RNA used was typically ³ 1.9. The quality of RNA was assessed by using the Agilent 2100 Bioanalyzer.
The probe labelling and hybridisation procedures were conducted by following the Affymetrix Technical Manual (Affymetrix, Santa Clara, CA). cDNA was synthesized from the total RNA by using Superscript ds-cDNA Synthesis Kit (Invitrogen Ltd, Paisley, U.K.) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. The cRNA was prepared and biotin-labelled by in vitro transcription by using BioArray High Yield RNA Transcript Labelling Kit (ENZO Biochemical). Labelled cRNA was fragmented by incubation at 940C for 35 min in the presence of 40 mM Tris acetate, pH 8.1, 100mM potassium acetate, and 30 mM magnesium acetate. The samples were tested with hybridization to GeneChip Test3 arrays and analysed by Agilent Bioanalyzer. Fragmented cRNA (15 mg) was hybridized 16h at 450C to an MG_U74Av2 array (Affymetrix). After hybridization, the gene chips were washed and stained with streptavidin-phycoerythrin by using a fluidics station (Affymetrix). The chips were scanned in an Agilent G2500A scanner. Affymetrix oligonucleotide microarrays utilize multiple perfect-match and mismatch oligonucleotides to determine expression levels, so Affymetrix GCOS software was used to scan, determine the presence and the average difference value, and assess signal intensity of each probe set. Chip fluorescence was normalized by scaling total chip fluorescence intensity to a common value of 100 prior to comparison, and a normalisation value was set at 1. Data and statistical analyses were performed with Genespring Ver 6.1 (Silicon Genetics, Redwood City, CA) bioinformatics algorithms.

source_name: mouse liver


title: HC_Hyb11_F124_15H8C_t90


title: HC_Hyb11_F249_13H8C_t90


title: Hepatic Reductase null N7


title: control mouse WT8


title: control mouse WT7


title: HUVEC 0,5hr IL1

description: Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4)  at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500. The given sample record represents the 0,5 hr time point of IL-1 stimulation.

source_name: Total RNA from IL-1 stimulated HUVEC (0,5hr)


title: HUVEC 0hr Ctl

description:
Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturer’s protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4)  at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500.
The given sample record represents the 0 hr (untreated) control measurement, which was finally used for pairwise comparison to each of the four time points of IL-1 stimulation.

source_name: Total RNA from unstimulated HUVEC


title: AJ_HF_s7_Week0

source_name: control, Cy3 dye, mixture of total RNA from liver, skeletal muscle, brain, heart, lung, testes, hypothalamus, brown adipose tissue, white adipose tissue, and kidney of C57 mice'


title: wt JRS4 cy3 vs mt JRS550 cy5 late exp (slide 4)

status: Public on Jul 14 2004

city: Hamilton

first: Frank

middle: R.

last: DeLeo

state: MT

address: 903 South 4th Street

department: Laboratory of Human Bacterial Pathogenesis

institute: National Institute of Allergy and Infectious Diseases

description:
Wild-type (JRS4) and irr mutant (JRS550) Group A Streptococcus (GAS) strains
Wild-type (JRS4) and irr mutant (JRS550) Group A Streptococcus (GAS) strains were cultured in Todd-Hewitt broth containing 0.5% yeast extract to early (OD600 0.35) and late (OD600 0.75) exponential phases of growth. Bacteria (2.5 x 109) were lysed with 700 µl RLT buffer (Qiagen, Valencia, CA) and the lysate was homogenized with an FP120 FastPrep system (Qbiogene Inc., Carlsbad, CA). Total RNA was isolated with RNeasy kits (Qiagen). First-strand cDNA synthesis to incorporate Cy3 and Cy5 labels, and tyramide signal amplification (TSA(tm)), was performed for RNA samples using a Micromax(tm) TSA(tm)  Labeling and Detection Kit (Perkin Elmer Life Sciences). cDNA labeled with Cy3 (mutant GAS strain) and Cy5 (wild-type GAS strain) were combined (50 µl total) and then diluted 1:1 with SlideHyb 3 (Ambion, Austin, TX). Samples were denatured at 95ºC for 2 min and incubated with a DNA microarray containing 1705 (of 1752) M1 GAS ORFs based on strain SF370, and unique M18 and M3 ORFs. PCR products derived from each ORF were printed onto CMT-GAPS Corning glass slides (Acton, MA) with a Chipwriter robotic arrayer (Bio-Rad, Hercules, CA) in at least quadruplicate. Hybridization of cDNA samples to microarray slides was conducted overnight in a 45ºC water bath. Slide signal was amplified with TSA (tm) and slides were scanned with a ScanArray 5000 instrument (PE Biosystems, San Diego, CA). Slides were normalized to equal fluorescence intensity against serial dilutions of MGAS8232 genomic DNA standards by adjusting laser power and/or photomultiplier gain. Spot location and array alignment were adjusted with QuantArray (PE Biosystems). To compare gene expression between the wild-type and irr mutant GAS strains, fold-changes for each gene were determined by the ratio of median fluorescence intensity of microarray spots derived from RNA samples of wild-type and irr mutant strains. Analysis of microarrays was done with GENESPRING software, version 4.2 (Silicon Genetics, Redwood City, CA). Microarray experiments were performed in duplicate. Slides 1 and 2 compare mutant and wild-type GAS strains during early exponential growth (OD600 0.35).  Slides 3 and 4 compare mutant and wild-type GAS strains during late exponential growth (OD600 0.75). Each microarray slide contained 4–12 spots per gene for a total of at least 8 spots for each gene.
Keywords = Human
Keywords = Neutrophils
Keywords = Bacterial
Keywords = Gene Regulation
Keywords = Inflammation

organism: Streptococcus pyogenes M1 GAS

source_name: wt JRS4

source_name: mt JRS550


title: CGEN12588692

status: Public on Jul 02 2004

city: Bar Harbor

first: Janet

last: Pereira

state: ME

address: 600 Main St.

department: Gene Expression Service

institute: The Jackson Laboratory

description:
Differential gene expression in four types of neoplasms originally arising in mice transgenic for c-Myc (FVB/N-Tg(WapMyc)212Bri) and selected through in vivo selection.
Mammary neoplasms arose in mice transgenic for c-Myc. These neoplasms were comprised of areas with different phenotypes. These phenotypes were: (1) glandular; (2) solid/comedo; and (3) spindloid. Through in vivo selection, we have generated 4 lines of tumors: (1) tumors composed exclusively of epithelial neoplastic cells arranged in a glandular pattern; (2) tumors composed of epithelial neoplastic cells arranged in a solid/comedo pattern and that were not associated with metastases to the lungs; (3): tumors composed of epithelial neoplastic cells arranged in a solid/comedo pattern and that were associated with metastases to the lungs; and (4) tumors composed of spindloid (=mesenchymal) neoplastic cells arranged in streams and bundles.
Tissue samples stored in RNAlater (Ambion, Austin, TX), were homogenized in TRIzol (Invitrogen, Carlsbad, CA). Total RNA was isolated by standard TRIzol methods according to the manufacturer’s protocols and quality was assessed using a 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA). Complementary cDNA was synthesized using 20 µg of each total RNA sample using the SuperScript Indirect cDNA Labeling System (Invitrogen).  The resulting amino-modified cDNA was labeled according to the manufacturer’s protocols using Cy3 or Cy5 fluorescent dyes (Amersham Biosciences, Piscataway, NJ). Labeled samples were hybridized in pairs using a dye-swap to the Universal Mouse Reference custom-printed oligonucleotide probe microarrays (Stratagene, La Jolla, CA; (7). The microarray probe set employed was the 22K Mouse Release 2.0 OligoLibrary (Compugen, Jamesburg, NJ and Sigma-Genosys, The Woodlands, TX). Hybridizations were performed at 42°C for 16-20 hours. Slides were washed and dried prior to scanning. Microarray images were obtained by scanning each slide at 10µm resolution in a GenePix 4000B scanner (Axon Instruments, Union City, CA). Image quantitation was performed using the associated software, GenePix ver. 4.0 and median intensity values (non-background subtracted) for each spot were used in subsequent data analysis.
Keywords = mammary, mouse, transgenic, epithelial to mesenchymal transition

source_name: TUMOR NAME GC03-643-A


title: CGEN12588943

source_name: TUMOR NAME GC03-669-A


title: wt JRS4 cy3 vs mt JRS550 cy5 early exp (slide1)


title: wt JRS4 cy3 vs mt JRS550 cy5 late exp (slide 3)


title: CGEN12588690


title: CGEN12588634

source_name: TUMOR NAME GC03-664-A


title: CGEN12588633

source_name: TUMOR NAME GC03-673-A


title: wt JRS4 cy3 vs mt JRS550 cy5 early exp (slide 2)


title: CGEN12588562

source_name: TUMOR NAME GC03-649-A


title: CGEN12588632

source_name: TUMOR NAME GC03-667-A


title: CGEN12588685


title: CGEN12588564

source_name: TUMOR NAME GC03-647-A


title: CGEN12588637


title: CGEN12588688

source_name: TUMOR NAME GC03-660-A


title: CGEN12588565

source_name: TUMOR NAME GC03-646-A


title: CGEN12588563


title: CGEN12588533

source_name: TUMOR NAME GC03-665-A


title: CGEN12588535

source_name: TUMOR NAME GC03-670-A


title: CGEN12585204

source_name: TUMOR NAME GC03-655-A


title: CGEN12588567


title: CGEN12588536


title: CGEN12585203

source_name: TUMOR NAME GC03-662-A


title: CGEN12588538

source_name: TUMOR NAME GC03-641-A


title: CGEN12588537


title: CGEN12585201


title: CGEN12585199


title: CGEN12585190

source_name: TUMOR NAME GC03-656-A


title: CGEN12585202


title: CGEN12588635


title: CGEN12585105

source_name: TUMOR NAME GC03-663-A


title: CGEN12585108


title: slide32 0 vs 0 hour-2

source_name: Control


title: CEA_SGF:AGQ032

status: Public on Jul 01 2004

city: Villejuif

first: Dalila

last: Sekkai

department: hematopoiesis and stem cell

country: France

institute: IGR

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AGQ

source_name: Reference 0h (Cy5)

source_name: Tet OFF 16h (Cy3)


title: CGEN12585103

source_name: TUMOR NAME GC03-653-A


title: CEA_SGF:AGQ029

description:
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Lot batch = CEA_SGF:AGQ

source_name: Tet OFF 48h (Cy5)

source_name: Reference 0h (Cy3)


title: CEA_SGF:AGQ033

description:
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AGQ

source_name: Tet OFF 24h (Cy5)


title: CGEN12585189


title: CEA_SGF:AGQ030

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AGQ

source_name: Tet OFF 48h (Cy3)


title: CGEN12568076

source_name: TUMOR NAME GC03-638-A


title: CEA_SGF:AGQ034

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AGQ

source_name: Tet OFF 24h (Cy3)


title: CGEN12585104

source_name: TUMOR NAME GC03-660-A


title: CGEN12564344


title: CGEN12567987

source_name: TUMOR NAME GC03-644-A


title: CGEN12567990

source_name: TUMOR NAME GC03-646-A


title: CEA_SGF:AGQ031

description:
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AGQ

source_name: Tet OFF 16h (Cy5)


title: CGEN12564342

source_name: TUMOR NAME GC03-639-A


title: CGEN12568077


title: CGEN12585107


title: CGEN12567986

source_name: TUMOR NAME GC03-640-A


title: CGEN12567985


title: CEA_SGF:AHU025

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU026

description:
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU027

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU019

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU015


title: CEA_SGF:AHU016

description:
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU014


title: CEA_SGF:AHU024

description:
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU011


title: CGEN12567989


title: CEA_SGF:AHU010


title: 6 (Cy3) - 17 (Cy5)

status: Public on Apr 28 2005

city: Columbia

first: Raja

middle: S

last: Settivari

state: MO

address: 920 E Campus Drive

department: ASRC

institute: University of Missouri

description: Rats (n=24) were fed ad libitum diets containing either ground endophyte-free (E-) seed or endophyte–infected (E+) seed for five days at thermoneutrality (21°C).  Rats (n=24) with intraperitonial transmitters were used with core temperature (Tc) and general activity measured every 5 minutes during the study. At treatment end, the liver was removed, weighed and frozen in liquid nitrogen. Twelve rats were selected for microarray experiments, based on degree of sensitivity to fescue toxicosis.  Degree of sensitivity was based on changes in relative liver weights, feed intake, average daily gain, and Tc from pretreatment levels. RNA was extracted from liver samples, converted to cDNA, and hybridized with the printed oligonucleotide slides.

source_name: RAT # 17

source_name: RAT # 6


title: CEA_SGF:AHU022

description:
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AFU012

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AFU

source_name: Reference 0h (Cy5)


title: CEA_SGF:AHU037

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
-LIF 48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 48h (-LIF 48h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU

source_name: Without LIF 48h (Cy3)


title: CEA_SGF:AFU015

description:
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AFU


title: CEA_SGF:AHU023

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Tet OFF48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 48h to allow expression of STAT3F (Tet OFF 48h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU012


title: CEA_SGF:AFU013

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Tet OFF24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 24h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AFU


title: TSC-1B

first: Myeong

last: Lee

department: Gladstone Institute of Virology and Immunology

institute: University of California at San Francisco

description:
Subpopulations of human fetal thymocyte and circulating naïve T cells were obtained through FACS sorting, including CD3-CD4+CD8- intrathymic T progenitor cells (ITTP), CD3intCD4+CD8+ "double positive" thymocytes (DP), CD3highCD4+CD8- "single positive" thymocytes (SP4), CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+), and CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+).
Keywords = Microarray
Keywords = gene expression
Keywords = thymocytes
Keywords = naïve CD4+ T cell
Keywords = recent thymic emigrants

source_name: Thymic Stromal Cells


title: CEA_SGF:AHU049

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
-LIF 48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 48h (-LIF 48h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: TSC-1A

description:
Description Subpopulations of human fetal thymocyte and circulating naïve T cells were obtained through FACS sorting, including CD3-CD4+CD8- intrathymic T progenitor cells (ITTP), CD3intCD4+CD8+ "double positive" thymocytes (DP), CD3highCD4+CD8- "single positive" thymocytes (SP4), CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+), and CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+).
Keywords = Microarray
Keywords = gene expression
Keywords = thymocytes
Keywords = naïve CD4+ T cell
Keywords = recent thymic emigrants


title: AB4-3B

source_name: CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+)


title: AB4-3A


title: TSC-2B


title: CEA_SGF:AFU014

description:
Tet OFF16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of LIF 1000U/ml but without tetracycline for 24h to allow expression of STAT3F (Tet OFF 16h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AFU

source_name: Reference 0h (Cy3)


title: AB4-2B


title: CB4-1B

source_name: CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+)


title: CEA_SGF:AHU047

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
-LIF 24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 24h (-LIF 24h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU

source_name: Without LIF 24h (Cy3)


title: CB4-2B


title: CB4-2A


title: CEA_SGF:AHU036

description:
-LIF 48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 48h (-LIF 48h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU

source_name: Without LIF 48h (Cy5)


title: AB4-2A


title: TSC-2A


title: AB4-1B


title: CB4-1A


title: AB4-1A


title: CEA_SGF:AHU043


title: SP4-3A

source_name: CD3highCD4+CD8- "single positive" thymocytes (SP4) cells from human fetal thymus


title: SP4-2A


title: SP4-3B


title: CEA_SGF:AHU041

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
-LIF 24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 24h (-LIF 24h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU046

description:
-LIF 24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 24h (-LIF 24h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Lot batch = CEA_SGF:AHU

source_name: Without LIF 24h (Cy5)


title: CEA_SGF:AHU035


title: SP4-1B


title: CEA_SGF:AHU040

description:
-LIF 24h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 24h (-LIF 24h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU034


title: SP4-1A


title: DP-2B

source_name: CD3intCD4+CD8+ "double positive" thymocytes (DP) cells from human fetal thymus


title: DP-2A


title: CGEN12585188

description:
Differential gene expression in four types of neoplasms originally arising in mice transgenic for c-Myc (FVB/N-Tg(WapMyc)212Bri) and selected through in vivo selection.
Mammary neoplasms arose in mice transgenic for c-Myc. These neoplasms were comprised of areas with different phenotypes. These phenotypes were: (1) glandular; (2) solid/comedo; and (3) spindloid. Through in vivo selection, we have generated 4 lines of tumors: (1) tumors composed exclusively of epithelial neoplastic cells arranged in a glandular pattern; (2) tumors composed of epithelial neoplastic cells arranged in a solid/comedo pattern and that were not associated with metastases to the lungs; (3): tumors composed of epithelial neoplastic cells arranged in a solid/comedo pattern and that were associated with metastases to the lungs; and (4) tumors composed of spindloid (=mesenchymal) neoplastic cells arranged in streams and bundles.
Tissue samples stored in RNAlater (Ambion, Austin, TX), were homogenized in TRIzol (Invitrogen, Carlsbad, CA). Total RNA was isolated by standard TRIzol methods according to the manufacturer’s protocols and quality was assessed using a 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA). Complementary cDNA was synthesized using 20 µg of each total RNA sample using the SuperScript Indirect cDNA Labeling System (Invitrogen).  The resulting amino-modified cDNA was labeled according to the manufacturer’s protocols using Cy3 or Cy5 fluorescent dyes (Amersham Biosciences, Piscataway, NJ). Labeled samples were hybridized in pairs using a dye-swap to the Universal Mouse Reference custom-printed oligonucleotide probe microarrays (Stratagene, La Jolla, CA; (7). The microarray probe set employed was the 22K Mouse Release 2.0 OligoLibrary (Compugen, Jamesburg, NJ and Sigma-Genosys, The Woodlands, TX). Hybridizations were performed at 42°C for 16-20 hours. Slides were washed and dried prior to scanning. Microarray images were obtained by scanning each slide at 10µm resolution in a GenePix 4000B scanner (Axon Instruments, Union City, CA). Image quantitation was performed using the associated software, GenePix ver. 4.0 and median intensity values (non-background subtracted) for each spot were used in subsequent data analysis.
Keywords = mammary, mouse, transgenic, epithelial to mesenchymal transition

source_name: TUMOR NAME GC03-656-A


title: DP-1A


title: ITTP-3B

source_name: CD34lowCD3-CD4+CD8- intrathymic T progenitor (ITTP) cells from human fetal thymus


title: DP-1B


title: ITTP-3A


title: ITTP-2B


title: ITTP-1B


title: ITTP-1A


title: ITTP-2A

source_name: source CD34lowCD3-CD4+CD8- intrathymic T progenitor (ITTP) cells from human fetal thymus


title: CEA_SGF:AHU045

description:
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
-LIF 16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 16h (-LIF 16h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU

source_name: Without LIF 16h (Cy3)


title: CEA_SGF:AHU033

description:
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
-LIF 16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 16h (-LIF 16h). Cells were then trypsinized and total RNA was extracted.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU044

description:
-LIF 16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 16h (-LIF 16h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Lot batch = CEA_SGF:AHU

source_name: Without LIF 16h (Cy5)


title: CEA_SGF:AHU039


title: CEA_SGF:AHU048

description:
-LIF 48h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 48h (-LIF 48h). Cells were then trypsinized and total RNA was extracted.
ARN ref 4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml).
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU038

description:
-LIF 16h ARN test  4x105 cells were plated in a 10 cm cell culture plate and cultured in the presence of tetracycline but without LIF for 16h (-LIF 16h). Cells were then trypsinized and total RNA was extracted.
ARN ref  4x105 cells were plated in a 10 cm cell culture plate and cultured under standard condition (+LIF 1000 U/ml, tetracycline 1µg/ml) for 48h.
Lot batch = CEA_SGF:AHU


title: CEA_SGF:AHU032


title: A404 SMC Differentiation Puromycin Replicate 4

status: Public on Jun 30 2004

city: Stanford

first: Joshua

middle: M.

last: Spin

address: 300 Pasteur Drive

institute: Stanford University

description:
Time point 4.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin. Cells were plated in 10-cm dishes and grown to near confluence. Medium in a set of plates was changed at time 0 to basal medium supplemented with 1 umol/L all trans-retinoic acid for 96 hours, followed by 48 hours of treatment with puromycin and harvesting (Puro group). RNA was extracted with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

RNA was purified and isolated using the Qiagen RNeasy Midi Kit Protocol. Ten ug of total RNA were primed with 2 ul of 100uM T16N2 DNA primer at 70 °C for 10 min, and reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase, and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3- or Cy5-dCTP and RNase inhibitor.  RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns. Oligoarray Control Targets and Hybridization Buffer (Agilent In Situ Hybrization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent EZ-Hybe chambers. After hybridization, slides were washed sequentially with 6× SSC/0.005% Triton X-102 and 0.1× SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, washed, and then scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.6.1.1).

source_name: Total RNA from P19 cells, clone A404, labeled with Cyanine-5 (red).

source_name: Total RNA from pooled whole mouse embryos e17.5, labeled with Cyanine-3 (green).


title: A404 SMC Differentiation Puromycin Replicate 3


title: A404 SMC Differentiation Puromycin Replicate 5


title: A404 SMC Differentiation Puromycin Replicate 6


title: A404 SMC Differentiation Puromycin Replicate 2


title: CEA_SGF:AHU042


title: A404 SMC Differentiation RA96 Replicate 4

description:
Time point 3.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin.
Cells were plated in 10-cm dishes and grown to near confluence. Medium in a set of plates at ~30% confluence was changed at time 0 to basal medium supplemented with 1 umol/L all trans-retinoic acid, and harvested after 96 hours of incubation (RA96 group). RNA was extracted with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

RNA was purified and isolated using the Qiagen RNeasy Midi Kit Protocol. Ten ug of total RNA were primed with 2 ul of 100uM T16N2 DNA primer at 70 °C for 10 min, and reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase, and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3- or Cy5-dCTP and RNase inhibitor.  RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns. Oligoarray Control Targets and Hybridization Buffer (Agilent In Situ Hybrization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent EZ-Hybe chambers. After hybridization, slides were washed sequentially with 6× SSC/0.005% Triton X-102 and 0.1× SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, washed, and then scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.6.1.1).


title: SP4-2B

description:
Subpopulations of human fetal thymocyte and circulating naïve T cells were obtained through FACS sorting, including CD3-CD4+CD8- intrathymic T progenitor cells (ITTP), CD3intCD4+CD8+ "double positive" thymocytes (DP), CD3highCD4+CD8- "single positive" thymocytes (SP4), CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+), and CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+).
Keywords = Microarray
Keywords = gene expression
Keywords = thymocytes
Keywords = naïve CD4+ T cell
Keywords = recent thymic emigrants


title: A404 SMC Differentiation Puromycin Replicate 1


title: A404 SMC Differentiation RA96 Replicate 2


title: A404 SMC Differentiation RA48 Replicate 2

description:
Time point 2.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin.
Cells were plated in 10-cm dishes and grown to near confluence. Medium in a set of plates at ~30% confluence was changed at time 0 to basal medium supplemented with 1 umol/L all trans-retinoic acid, and harvested after 48 hours of incubation (RA48 group). RNA was extracted with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

RNA was purified and isolated using the Qiagen RNeasy Midi Kit Protocol. Ten ug of total RNA were primed with 2 ul of 100uM T16N2 DNA primer at 70 °C for 10 min, and reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase, and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3- or Cy5-dCTP and RNase inhibitor.  RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns. Oligoarray Control Targets and Hybridization Buffer (Agilent In Situ Hybrization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent EZ-Hybe chambers. After hybridization, slides were washed sequentially with 6× SSC/0.005% Triton X-102 and 0.1× SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, washed, and then scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.6.1.1).


title: PC3 4 HrTime Point

status: Public on Jun 24 2004

city: Gaithersburg

first: Jeff

last: Kiefer

state: MD

address: 20 Firstfield Rd,  Suite 110

department: CDDL

institute: Translational Genomics Research Institute (TGen)

description: PC3 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 4 hour time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: PC3 4 Hr Time Point Emetine Treated

source_name: PC3 4 Hr Time Point Un-Treated


title: A404 SMC Differentiation RA96 Replicate 1