title: C57_HF_s38_Week4

description:
C57 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: C57_HF_s45_Week5

status: Public on Jul 31 2004

type: RNA

city: Cambridge

first: Jatin

last: Misra

state: MA

country: USA

institute: MIT

description:
C57 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).

molecule: total RNA

organism: Mus musculus

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

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: C57_HF_s43_Week5


title: C57_HF_s41_Week5


title: C57_HF_s40_Week4


title: C57_HF_s36_Week4


title: C57_HF_s34_Week4


title: C57_HF_s52_Week6

description:
C57 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: C57_HF_s31_Week4


title: C57_HF_s30_Week3

description:
C57 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: C57_HF_s42_Week5


title: C57_HF_s27_Week3


title: C57_HF_s28_Week3


title: C57_HF_s37_Week4


title: C57_HF_s32_Week4


title: C57_HF_s33_Week4


title: C57_HF_s35_Week4


title: C57_HF_s29_Week3


title: C57_HF_s39_Week4


title: C57_HF_s26_Week3


title: C57_HF_s24_Week3


title: C57_HF_s21_Week3


title: C57_HF_s23_Week3


title: C57_HF_s47_Week5


title: C57_LF_s8_Week0

description:
C57 mouse, total liver RNA, low-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, low-fat diet, Week0


title: C57_HF_s16_Week1

description:
C57 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: C57_HF_s7_Week0

description:
C57 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: C57_HF_s12_Week1


title: C57_HF_s15_Week1


title: C57_HF_s17_Week1


title: C57_HF_s22_Week3


title: C57_HF_s14_Week1


title: C57_HF_s19_Week1


title: C57_HF_s2_Week0


title: C57_HF_s3_Week0


title: C57_HF_s6_Week0


title: AJ_LF_s108_Week11

description:
AJ mouse, total liver RNA, low-fat diet, Week11
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, Week11


title: C57_HF_s4_Week0


title: C57_HF_s1_Week0


title: C57_HF_s10_Week0


title: C57_HF_s13_Week1


title: C57_HF_s20_Week1


title: C57_HF_s11_Week1


title: C57_HF_s9_Week0


title: C57_HF_s5_Week0


title: C57_HF_s18_Week1


title: C57_HF_s8_Week0


title: AJ_LF_s107_Week11


title: AJ_LF_s106_Week11


title: AJ_LF_s99_Week10

description:
AJ mouse, total liver RNA, low-fat diet, Week10
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, Week10


title: AJ_LF_s100_Week11


title: AJ_LF_s101_Week11


title: AJ_LF_s95_Week10


title: AJ_LF_s105_Week11


title: AJ_LF_s96_Week10


title: AJ_LF_s104_Week11


title: AJ_LF_s91_Week10


title: AJ_LF_s98_Week10


title: AJ_LF_s93_Week10


title: AJ_LF_s97_Week10


title: AJ_LF_s85_Week9

description:
AJ mouse, total liver RNA, low-fat diet, Week9
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, Week9


title: AJ_LF_s87_Week9


title: AJ_LF_s88_Week9


title: AJ_LF_s90_Week10


title: AJ_LF_s89_Week9


title: AJ_LF_s82_Week9


title: AJ_LF_s92_Week10


title: AJ_LF_s86_Week9


title: AJ_LF_s80_Week9


title: AJ_LF_s103_Week11


title: AJ_LF_s81_Week9


title: AJ_LF_s83_Week9


title: AJ_LF_s84_Week9


title: AJ_LF_s79_Week8

description:
AJ mouse, total liver RNA, low-fat diet, Week8
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, Week8


title: AJ_LF_s102_Week11


title: AJ_LF_s77_Week8


title: AJ_LF_s94_Week10


title: AJ_LF_s75_Week8


title: AJ_LF_s78_Week8


title: AJ_LF_s74_Week8


title: AJ_LF_s73_Week8


title: AJ_LF_s70_Week8


title: AJ_LF_s76_Week8


title: AJ_LF_s69_Week7

description:
AJ mouse, total liver RNA, low-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, low-fat diet, Week7


title: AJ_LF_s68_Week7


title: AJ_LF_s61_Week7


title: AJ_LF_s66_Week7


title: AJ_LF_s59_Week6

description:
AJ mouse, total liver RNA, low-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, low-fat diet, Week6


title: AJ_LF_s62_Week7


title: AJ_LF_s64_Week7


title: C57_HF_s25_Week3


title: AJ_LF_s57_Week6


title: AJ_LF_s56_Week6


title: AJ_LF_s65_Week7


title: AJ_LF_s55_Week6


title: AJ_LF_s72_Week8


title: AJ_LF_s71_Week8


title: AJ_LF_s50_Week6


title: AJ_LF_s52_Week6


title: AJ_LF_s63_Week7


title: AJ_LF_s49_Week5

description:
AJ mouse, total liver RNA, low-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, low-fat diet, Week5


title: AJ_LF_s51_Week6


title: AJ_LF_s48_Week5


title: AJ_LF_s47_Week5


title: AJ_LF_s44_Week5


title: AJ_LF_s42_Week5


title: AJ_LF_s46_Week5


title: AJ_LF_s45_Week5


title: AJ_LF_s40_Week5


title: AJ_LF_s54_Week6


title: AJ_LF_s67_Week7


title: AJ_LF_s38_Week4

description:
AJ mouse, total liver RNA, low-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, low-fat diet, Week4


title: AJ_LF_s60_Week7


title: AJ_LF_s41_Week5


title: AJ_LF_s37_Week4


title: AJ_LF_s33_Week4


title: AJ_LF_s39_Week4


title: AJ_LF_s35_Week4


title: AJ_LF_s34_Week4


title: AJ_LF_s32_Week4


title: AJ_LF_s36_Week4


title: AJ_LF_s31_Week4


title: AJ_LF_s53_Week6


title: AJ_LF_s30_Week4


title: AJ_LF_s29_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_LF_s27_Week3


title: AJ_LF_s28_Week3


title: AJ_LF_s23_Week3


title: AJ_LF_s26_Week3


title: AJ_LF_s25_Week3


title: AJ_LF_s24_Week3


title: AJ_LF_s18_Week1

description:
AJ mouse, total liver RNA, low-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, low-fat diet, Week1


title: AJ_LF_s22_Week3


title: AJ_LF_s20_Week3


title: AJ_LF_s13_Week1


title: AJ_LF_s15_Week1


title: AJ_LF_s16_Week1


title: AJ_LF_s14_Week1


title: AJ_LF_s6_Week0

description:
AJ mouse, total liver RNA, low-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).


title: AJ_LF_s11_Week1


title: AJ_LF_s19_Week1


title: AJ_LF_s17_Week1


title: AJ_LF_s8_Week0


title: AJ_LF_s12_Week1


title: AJ_LF_s9_Week0


title: AJ_LF_s10_Week1


title: AJ_LF_s7_Week0


title: AJ_LF_s4_Week0


title: AJ_LF_s3_Week0


title: AJ_LF_s5_Week0


title: AJ_HF_s108_Week11

description:
AJ mouse, total liver RNA, high-fat diet, Week11
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, Week11


title: AJ_HF_s110_Week11

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_LF_s2_Week0


title: AJ_HF_s107_Week11


title: AJ_LF_s1_Week0


title: AJ_LF_s58_Week6


title: AJ_HF_s109_Week11


title: AJ_HF_s105_Week11


title: AJ_HF_s104_Week11


title: AJ_HF_s103_Week11


title: AJ_HF_s102_Week11


title: AJ_HF_s106_Week11


title: AJ_HF_s101_Week11


title: AJ_HF_s98_Week10

description:
AJ mouse, total liver RNA, high-fat diet, Week10
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, Week10


title: AJ_HF_s99_Week10


title: AJ_HF_s97_Week10


title: AJ_HF_s94_Week10


title: AJ_HF_s95_Week10


title: AJ_HF_s100_Week10


title: AJ_HF_s93_Week10


title: AJ_HF_s96_Week10


title: AJ_HF_s92_Week10


title: AJ_HF_s91_Week10


title: AJ_HF_s89_Week9

description:
AJ mouse, total liver RNA, high-fat diet, Week9
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, Week9


title: AJ_HF_s90_Week9


title: AJ_LF_s43_Week5


title: AJ_HF_s88_Week9


title: AJ_HF_s83_Week9


title: AJ_HF_s78_Week8

description:
AJ mouse, total liver RNA, high-fat diet, Week8
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, Week8


title: AJ_HF_s82_Week9


title: AJ_HF_s84_Week9


title: AJ_HF_s80_Week8


title: AJ_HF_s79_Week8


title: AJ_HF_s77_Week8


title: AJ_HF_s87_Week9


title: AJ_HF_s85_Week9


title: AJ_HF_s75_Week8


title: AJ_HF_s76_Week8


title: AJ_HF_s73_Week8


title: AJ_HF_s74_Week8


title: AJ_HF_s81_Week9


title: AJ_HF_s71_Week8


title: AJ_HF_s72_Week8