title: yuccaBR1

status: Public on Aug 31 2004

city: La Jolla

first: Jennifer

last: Nemhauser

department: PBIO

institute: Salk Institute

description: Seedlings were immersed in 1 µM brassinolide in one-half-strength Murashige Minimal Organics Medium (Invitrogen, Carlsbad, CA) for 2.5 hours

organism: Arabidopsis thaliana

source_name: Ten-day old light-grown yucca seedlings


title: Control 1b Chip A

status: Public on Sep 01 2004

type: RNA

city: El Segundo

first: Anton

last: Petrov

state: CA

country: USA

institute: BioDiscovery Inc

description: At confluence, keratinocytes from the DK-7 cell line were exposed to 100 IU/ml of IFN-g in serum free medium. After 24 hrs, RNA was extracted using the Qiagen Rneasy Mini Kit (Qiagen SA, Cedex, France). All the samples were monitored by agarose gel and with the Agilent 2100 Bioanalyser (Agilent Biotechnologies, Germany) and consistently demonstrated high-quality RNA (28S/18S ratio approximately 2, but always less than 3). According to Affymetrix protocol, 5 µg total RNA was the starting material for all individual samples. In general, total RNA was converted to biotinylated cRNA, hybridized in the Affymetrix probe array cartridge, stained, and then quantified. First and second strand cDNA synthesis was performed using the SuperScript Choice System (Invitrogen AG, Basel, Switzerland), according to manufacturer instructions, but using an oligo-dT primer containing a T7 RNA polymerase binding site. Labeled cRNA was prepared with the RNA Transcript Labeling kit (Enzo Biochem Inc., NY). Biotinylated CTP and UTP were used together with unlabeled NTPs in the reaction, and unincorporated nucleotides were removed with GeneChipâ Cleanup Module (Affymetrix, Inc., Santa Clara, CA) . cRNA (20 µg) was fragmented at 94 °C for 35 min in buffer containing 200 mM Tris-acetate pH 8.1, 500 mM KOAc, and 150 mM MgOAc. Prior to hybridization, fragmented cRNA in hybridization mix ( Buffer containing 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20, 0.5 ng/ml BSA, 0.1 ng/ml herring sperm and Affymetrix controls ), was heated to 95 °C for 5 min, cooled to 45 °C and loaded onto an Affymetrix probe array cartridge. The probe array was incubated for 16 hrs at 45 °C at constant rotation (60 rpm), then exposed to Affymetrix washing and staining protocol. This protocol includes: · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · one wash with stringent buffer ( 100 mM MES, 0.1 M NaCl, 0.01 % Tween 20 ) · First stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · Second stain with 3 mg/ml of biotinylated anti-streptavidin + 0.2 mg/ml of IgG in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · Third stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) Probe arrays were scanned at 488 nm using an Argon-ion Laser (made for Affymetrix by Agilent). Readings from the quantitative scanning were analyzed with Affymetrix Gene Expression Analysis Software (MAS 5.0). This experiment was comparing control versus IFN-g treated skin cells (one design factor on two levels). To evaluate the biological and the experimental variability, the following experimental design was planned as shown in Figure 1: "Control" and "IFN-g stimulated" keratinocytes were cultured in triplicate using three individual petri Æ10 culture dishes and RNA was independently extracted from each cultured replicate. For each RNA sample, cRNA synthesis was performed in triplicate and each cRNA pool was hybridized to an Affymetrix U133 Gene Chip.

molecule: total RNA

organism: Homo sapiens

source_name: keratinocytes from DK-7


title: Control 2b Chip A


title: Control 2c Chip A


title: IFN 5c Chip A


title: IFN 5b Chip A


title: IFN 6a Chip A


title: wt(zm)IAA2

description: Seedlings were immersed in 10 µM indole-3-acetic acid (IAA) in water for 2 hours

source_name: Five-day old light-grown Columbia wild type (Col-0) seedlings


title: Control 4a Chip A


title: yuccaBR2


title: WTBR1

source_name: Ten-day old light-grown Columbia wild type (Col-0) seedlings


title: Control 1a Chip A


title: WTBR2


title: wt(zm)IAA1


title: Control 2a Chip A


title: 360min_Rep3

status: Public on Aug 24 2004

city: Stillwater

first: Robert

last: Burnap

state: OK

institute: Oklahoma State University

description:
Synechocystis 6803 cells were grown photoautotrophically at 32°C in buffered BG-11 and bubbled with 3% CO2. A relatively mild Ci stress was applied by switching the aeration from 3% CO2 to air alone. After incubation for the designated time, a 100-ml aliquot of culture was immediately combined with an equal volume of ice-cold mixture of phenol and ethanol (1:10, w/v) in an ice bath. The resultant cells were collected by centrifugation at 1000 x g for 10 min at 4'C. Total RNA was isolated with RNeasy Midi Kit (Qiagen, Valencia, CA) and further treated with the DNA-free kit (Ambion, Austin, TX). Fluorescently labeled cDNA was produced via a two-step indirect procedure involving cDNA synthesis from 16 ug of total RNA in a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide, followed by the second step involving chemical coupling of fluorescent dye to the introduced amino moieties of the synthesized cDNA. Labeled cDNA were adjusted to 14.75 ul, and the remainder of the hybridization components containing 2.5 ul of 10 ug ul-1 salmon sperm DNA, 8.75 ul of 20x SSC, 0.25 ul of 10% SDS, and 8.75 ul of formamide were added. The mixture was then heated for 2 min at 99'C and maintained at 42'C until hybridization. Hybridizations were preformed in a static incubator at 42'C for 12-16 h then washed by placing in a 250-ml solution of 2x SSC and 0.1% SDS at 42'C for 5 min with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a 250-ml solution of 0.1x SSC and 0.1% SDS, incubated for 10 min at room temperature with gentle agitation, and washed five additional times in 0.1x SSC for 1 min at room temperature. Hybridization signals from the microarray were quantified using GenePix Pro 4.1 (Axon Instruments, Union City, CA). The quality control procedures were conducted in the image analysis software, and then data were saved to Acuity 3.1 (Axon Instruments).
Keywords = Synechocystis, PCC 6803

organism: Synechocystis sp. PCC 6803

source_name: 360 min post inorganic carbon limitation

source_name: 0 min inorganic carbon limitation


title: 720min_Rep2

source_name: 720 min post inorganic carbon limitation


title: 360min_Rep1


title: 720min_Rep1


title: 200min_Rep4

source_name: 200 min post inorganic carbon limitation


title: 60min_Rep3

source_name: 60 min post inorganic carbon limitation


title: 200min_Rep3


title: 180min_Rep2

source_name: 180 min post inorganic carbon limitation


title: 200min_Rep1


title: 180min_Rep3


title: 200min_Rep2


title: 180min_Rep1


title: 60min_Rep2


title: Hydrocortisone_CM_3

status: Public on Aug 18 2004

city: San Antonio

first: Phillip

last: Bowman

state: TX

address: 3400 Rawley E. Chambers Ave

institute: Institute of Surgical Research

description: Pooled first passage cryopreserved, human umbilical vein endothelial cells (HUVEC) were obtained from Cascade Biologics (Portland, OR), thawed, and cultured according to the supplier’s recommendations in Medium 200. After thawing, cells were seeded onto 0.2% gelatin coated tissue culture plates and cultivated under 95% air, 5% CO2 at 37 oC.

source_name: HUVEC treated with 1 µM hydrocortisone for 1 hr and then with a mixture of cytokines (CM) for 4 hrs

source_name: Human universal reference RNA (Stratagene, La Jolla, CA)


title: Hydrocortisone_LPS_4

source_name: HUVEC treated with 1 µM hydrocortisone for 1 hr and then with 100 ng/ml LPS for 4 hrs


title: Hydrocortisone_LPS_3


title: 60min_Rep1


title: Hydrocortisone_CM_2


title: 360min_Rep2


title: Hydrocortisone_CM_4


title: Hydrocortisone_LPS_1


title: Hydrocortisone_Ctl_4

source_name: HUVEC treated with 1 µM hydrocortisone for 5 hrs


title: Hydrocortisone_Ctl_1


title: Hydrocortisone_Ctl_2


title: Hydrocortisone_Ctl_3


title: Hydrocortisone_LPS_2


title: 720min_Rep3


title: Estradiol_CM_2

source_name: HUVEC pretreated with 1 µM 17β-estradiol for 1 hr and then with a mixture of cytokines (CM) for 4 hrs


title: Estradiol_LPS_2

source_name: HUVEC pretreated with 1 µM 17β-estradiol for 1 hr and then 100 ng/ml LPS for 4 hrs


title: Estradiol_CM_3


title: Estradiol_LPS_4


title: Estradiol_CM_1


title: TAcKLE RT 40 µg BR (ID393)

status: Public on Aug 13 2004

city: Heidelberg

first: Joerg

last: Schlingemann

address: Technologiepark III, Im Neuenheimer Feld 280

department: Molecular Biology (B060)

country: Germany

institute: German Cancer Research Center (DKFZ)

description:
Labeling was achieved by reverse transcription with Cy-dUTPs. Hybridization was carried out at 37 °C in UltraHyb buffer (Ambion) on a GeneTAC hybridization station (Genomic Solutions).
Keywords = amplification
Keywords = labeling
Keywords = protocol
Keywords = spotted
Keywords = human
Keywords = long oligonucleotide
Keywords = oligo
Keywords = 70mer
Keywords = Operon
Lot batch =

source_name: 40 µg total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)

source_name: 40 µg universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000)


title: Estradiol_LPS_3


title: TAcKLE RT 40 µg BR (ID392)


title: IFN 6c Chip A

source_name: keratinocytes from DK-7


title: TAcKLE RT 40 µg RR (ID391)


title: Naive B6.2.16 CD8 T cells

status: Public on Aug 05 2004

city: Chicago

first: Philip

middle: George

last: Ashton-Rickardt

state: IL

address: 924 E.57th Street

department: Pathology

institute: The University of Chicago

description: Naive B6.2.16 CD8 T cells (>85% pure) were isolated from the lymph nodes (LN) of female RAG1-deficient B6.2.16 mice. Sample proRNA from naïve B6.2.16 CD8 T cells was isolated using Trizol(r) Reagent and used to make cRNA for hybridization according to Affymatrix protocols.

organism: Mus musculus

source_name: Naive B6.2.16 CD8 T cells


title: TAcKLE 2 ng RR (ID388)

description:
aRNA produced by T7 amplification was reversely transcribed and subsequently labeled using Klenow fragment and Cy-dUTPs. Hybridization was carried out at 37 °C in UltraHyb buffer (Ambion) on a GeneTAC hybridization station (Genomic Solutions).
Keywords = amplification
Keywords = labeling
Keywords = protocol
Keywords = spotted
Keywords = human
Keywords = long oligonucleotide
Keywords = oligo
Keywords = 70mer
Keywords = Operon
Lot batch =

source_name: 2 ng universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000)


title: TAcKLE 20 ng BR (ID385)

source_name: 20 ng total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)

source_name: 20 ng universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000)


title: Human activated CD56+CD16+ NK subset - replicate (B)

status: Public on Aug 02 2004

city: Jerusalem

first: Ofer

last: Mandelboim

department: The Lautenberg Center for General and Tumor Immunology

country: Israel

institute: The Hebrew University of Jerusalem

description: Total cellular RNA from polyclonal CD56+CD16+ in vitro activated NK subset (100U/ml IL-2 + 1ng/ml PHA)was pooled from 9 different donors was isolated and subjected to clean up protocol with RNeasy mini kits (Qiagen), according to the manufacturer’s specifications. The Quality of total RNA was assessed using an Agilent Bioanalyzer. First and second strand cDNA was prepared from 0.5µg RNA template, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was fragmented to uniform sizes (~100nt) CodeLinkTM Uniset Human 20K I Bioarrays (Amersham Biosceinces) were hybridized with each prepared cRNA target in duplicates and stained with Cy5-streptavidin and subsequently washed according to manufacturer’s instructions, and then scanned on an Axon GenePix 4000B scanner. Raw data files were obtained after analysis of scanned images with CodeLinkTM expression software (Amersham Biosciences).

source_name: Pooled purified activated CD56+CD16+ NK subsets from 9 healthy donors


title: Memory B6.2.16 CD8 T cell

description: Naive B6.2.16 CD8 T cells (>85% pure) were isolated from the lymph nodes (LN) of female RAG1-deficient B6.2.16 mice.  B6.2.16 CD8 T cell effectors were generated by culturing splenocytes from female RAG1-deficient B6.2.16 mice with HY peptide for 4 d and adoptively transferred into female RAG1-deficient mice.  After 200 d, memory B6.2.16 CD8 T cells (>80% pure) were recovered from the spleens and LNs by magnetic bead sorting. Sample proRNA from naïve and memoryB6.2.16 CD8 T cells was isolated using Trizol(r) Reagent and used to make cRNA for hybridization according to Affymatrix protocols.

source_name: Memory B6.2.16 CD8 T cell


title: TAcKLE 2 ng RB (ID375)

source_name: 2 ng total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)


title: IFN 8b Chip A


title: TAcKLE RT 40 µg RB (ID373)


title: TAcKLE 2 ng BR (ID371)


title: Memory  B6.2.16 CD8 T cell

source_name: Memory  B6.2.16 CD8 T cell


title: TAcKLE 2000 ng RR (ID382)

source_name: 2000 ng universal human reference total RNA, pooled from 10 cell lines representing distinct tissues (Stratagene #740000)


title: Human activated CD56+CD16+ NK subset - replicate (A)


title: TAcKLE 2000 ng BR (ID379)

source_name: 2000 ng total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)


title: TAcKLE 200 ng RB (ID377)

source_name: 200 ng universal human reference total RNA, pooled from 10 human cell lines representing distinct tissues (Stratagene #740000)

source_name: 200 ng total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)


title: TAcKLE 20 ng RB (ID383)


title: Figure_2A_VEGF_8

status: Public on Sep 13 2005

city: Seattle

first: Amit

middle: V

last: Kulkarni

state: WA

address: 401 Terry Ave N

department: Genomics

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

description: human dermal microvascular endothelial cell RNA

data_processing: LogRatio of channels

source_name: HDMVEC_24h VEGF_8

tag: HDMVEC_24h VEGF_8


title: TAcKLE 200 ng BR (ID381)


title: TAcKLE 2000 ng BR (ID380)


title: TAcKLE 20 ng RR (ID370)


title: TAcKLE 20 ng BR (ID365)


title: TAcKLE 200 ng RR (ID366)


title: TAcKLE 2 ng BR (ID372)


title: Human CD56brightCD16- NK subset - replicate (B)

description: Total cellular RNA from CD56 brightCD16- NK subset was pooled from 9 different donors was isolated and subjected to clean up protocol with RNeasy mini kits (Qiagen), according to the manufacturer’s specifications. The Quality of total RNA was assessed using an Agilent Bioanalyzer. First and second strand cDNA was prepared from 0.5µg RNA template, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was fragmented to uniform sizes (~100nt) CodeLinkTM Uniset Human 20K I Bioarrays (Amersham Biosceinces) were hybridized with each prepared cRNA target in duplicates and stained with Cy5-streptavidin and subsequently washed according to manufacturer’s instructions, and then scanned on an Axon GenePix 4000B scanner. Raw data files were obtained after analysis of scanned images with CodeLinkTM expression software (Amersham Biosciences).

source_name: Pooled purified CD56brightCD16- NK subsets from 9 healthy donors


title: Human CD56dimCD16+ NK subset - replicate (B)

description: Total cellular RNA from CD56 dimCD16+ NK subset was pooled from 9 different donors was isolated and subjected to clean up protocol with RNeasy mini kits (Qiagen), according to the manufacturer’s specifications. The Quality of total RNA was assessed using an Agilent Bioanalyzer. First and second strand cDNA was prepared from 0.5µg RNA template, and the cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates. The biotinylated cRNA was fragmented to uniform sizes (~100nt) CodeLinkTM Uniset Human 20K I Bioarrays (Amersham Biosceinces) were hybridized with each prepared cRNA target in duplicates and stained with Cy5-streptavidin and subsequently washed according to manufacturer’s instructions, and then scanned on an Axon GenePix 4000B scanner. Raw data files were obtained after analysis of scanned images with CodeLinkTM expression software (Amersham Biosciences).

source_name: Pooled purified CD56dimCD16+ NK subsets from 9 healthy donors


title: Human CD56brightCD16- NK subset - replicate (A)


title: Human CD56dimCD16+ NK subset - replicate (A)


title: TAcKLE 200 ng BR (ID378)


title: C57_LF_s103_Week11

status: Public on Jul 31 2004

city: Cambridge

first: Jatin

last: Misra

state: MA

institute: MIT

description:
C57 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

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_LF_s106_Week11


title: C57_LF_s98_Week10

description:
C57 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: C57_LF_s109_Week11


title: C57_LF_s100_Week10


title: C57_LF_s104_Week11


title: C57_LF_s101_Week11


title: C57_LF_s92_Week10


title: C57_LF_s91_Week10


title: C57_LF_s94_Week10


title: C57_LF_s96_Week10


title: C57_LF_s108_Week11


title: C57_LF_s95_Week10


title: C57_LF_s99_Week10


title: Estradiol_CM_4

source_name: HUVEC pretreated with 1 µM 17β-estradiol for 1 hr and then with a mixture of cytokines (CM) for 4 hrs

source_name: Human universal reference RNA (Stratagene, La Jolla, CA)


title: C57_LF_s102_Week11


title: C57_LF_s93_Week10


title: C57_LF_s97_Week10


title: C57_LF_s87_Week9

description:
C57 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: C57_LF_s88_Week9


title: C57_LF_s90_Week9


title: C57_LF_s86_Week9


title: C57_LF_s105_Week11


title: C57_LF_s107_Week11


title: C57_LF_s89_Week9


title: C57_LF_s85_Week9


title: C57_LF_s83_Week9


title: C57_LF_s80_Week8

description:
C57 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: C57_LF_s82_Week9


title: C57_LF_s84_Week9


title: C57_LF_s77_Week8


title: Hydrocortisone_CM_1

source_name: HUVEC treated with 1 µM hydrocortisone for 1 hr and then with a mixture of cytokines (CM) for 4 hrs


title: C57_LF_s81_Week9


title: C57_LF_s75_Week8


title: C57_LF_s79_Week8


title: C57_LF_s74_Week8


title: C57_LF_s73_Week8


title: C57_LF_s68_Week7

description:
C57 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: C57_LF_s78_Week8


title: C57_LF_s72_Week8


title: C57_LF_s65_Week7


title: C57_LF_s71_Week8


title: C57_LF_s69_Week7


title: C57_LF_s67_Week7


title: C57_LF_s66_Week7


title: C57_LF_s61_Week7


title: C57_LF_s63_Week7


title: C57_LF_s62_Week7


title: C57_LF_s64_Week7


title: C57_LF_s70_Week7


title: C57_LF_s58_Week6

description:
C57 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: C57_LF_s53_Week6


title: C57_LF_s52_Week6


title: C57_LF_s49_Week5

description:
C57 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: C57_LF_s60_Week6


title: C57_LF_s50_Week5


title: C57_LF_s59_Week6


title: C57_LF_s57_Week6


title: C57_LF_s44_Week5


title: C57_LF_s56_Week6


title: C57_LF_s36_Week4

description:
C57 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: C57_LF_s46_Week5


title: C57_LF_s43_Week5


title: C57_LF_s37_Week4


title: C57_LF_s41_Week5


title: C57_LF_s51_Week6


title: C57_LF_s48_Week5


title: C57_LF_s45_Week5


title: C57_LF_s54_Week6


title: C57_LF_s38_Week4


title: C57_LF_s35_Week4


title: C57_LF_s31_Week4


title: C57_LF_s29_Week3

description:
C57 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: C57_LF_s40_Week4


title: TAcKLE 2000 ng RB (ID389)

source_name: 2000 ng universal human reference total RNA, pooled from 10 cell lines representing distinct tissues (Stratagene #740000)

source_name: 2000 ng total RNA extracted from normal breast tissue of a 56 year old female body (Stratagene #735044)


title: C57_LF_s39_Week4


title: C57_LF_s30_Week3


title: C57_LF_s32_Week4


title: C57_LF_s47_Week5


title: C57_LF_s55_Week6


title: C57_LF_s42_Week5


title: C57_LF_s76_Week8


title: C57_LF_s24_Week3


title: C57_LF_s20_Week1

description:
C57 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: C57_LF_s23_Week3


title: C57_LF_s22_Week3


title: C57_LF_s11_Week1


title: C57_LF_s27_Week3


title: C57_LF_s16_Week1


title: C57_LF_s28_Week3


title: C57_LF_s14_Week1


title: C57_LF_s21_Week3


title: C57_LF_s15_Week1


title: C57_LF_s34_Week4


title: C57_LF_s25_Week3


title: C57_LF_s17_Week1


title: C57_LF_s9_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_LF_s7_Week0


title: C57_LF_s6_Week0


title: C57_LF_s12_Week1


title: C57_LF_s10_Week0


title: C57_LF_s19_Week1


title: C57_LF_s13_Week1


title: C57_LF_s26_Week3


title: C57_LF_s18_Week1


title: C57_LF_s2_Week0


title: C57_HF_s107_Week11

description:
C57 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: C57_LF_s4_Week0

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


title: C57_LF_s3_Week0


title: C57_HF_s105_Week11


title: C57_HF_s108_Week11


title: C57_HF_s100_Week11


title: C57_HF_s98_Week10

description:
C57 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: C57_HF_s92_Week10


title: C57_HF_s94_Week10


title: C57_HF_s106_Week11


title: C57_HF_s89_Week10


title: C57_LF_s1_Week0


title: C57_HF_s102_Week11


title: C57_HF_s103_Week11


title: C57_HF_s91_Week10


title: C57_HF_s101_Week11


title: C57_HF_s95_Week10


title: C57_HF_s88_Week9

description:
C57 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: C57_HF_s99_Week11


title: C57_HF_s90_Week10


title: C57_HF_s93_Week10


title: C57_HF_s97_Week10


title: C57_HF_s96_Week10


title: C57_HF_s81_Week9


title: C57_HF_s73_Week8

description:
C57 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: C57_HF_s87_Week9


title: C57_HF_s79_Week8


title: C57_HF_s83_Week9


title: C57_HF_s85_Week9


title: C57_HF_s82_Week9


title: C57_HF_s80_Week8


title: C57_HF_s70_Week7

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

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


title: C57_HF_s84_Week9


title: C57_HF_s86_Week9


title: C57_HF_s75_Week8


title: C57_LF_s33_Week4


title: C57_HF_s72_Week8


title: C57_HF_s77_Week8


title: C57_HF_s74_Week8


title: C57_HF_s78_Week8


title: C57_HF_s64_Week7


title: C57_LF_s5_Week0


title: C57_HF_s60_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_s59_Week6


title: C57_HF_s71_Week8


title: C57_HF_s65_Week7


title: C57_HF_s67_Week7


title: C57_HF_s63_Week7


title: C57_HF_s58_Week6


title: C57_HF_s51_Week6


title: C57_HF_s49_Week5

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).

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


title: C57_HF_s48_Week5


title: C57_HF_s69_Week7


title: C57_HF_s56_Week6


title: C57_HF_s62_Week7


title: C57_HF_s66_Week7


title: C57_HF_s57_Week6


title: C57_HF_s61_Week7


title: C57_HF_s53_Week6


title: C57_HF_s68_Week7


title: C57_HF_s55_Week6


title: C57_HF_s76_Week8


title: C57_HF_s104_Week11


title: C57_HF_s54_Week6


title: C57_HF_s50_Week5


title: C57_HF_s44_Week5


title: C57_HF_s46_Week5