title: LNCaP 2 HR Time Point

description: LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 2 hour time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: LNCaP 2 HR Time Point Emetine Treated

source_name: LNCaP 2 HR Time Point Un-Treated


title: PC3 30' Time Point

status: Public on Jun 24 2004

type: RNA

city: Gaithersburg

first: Jeff

last: Kiefer

state: MD

address: 20 Firstfield Rd,  Suite 110

department: CDDL

country: USA

institute: Translational Genomics Research Institute (TGen)

description: PC3 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 30 min time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

molecule: total RNA

organism: Homo sapiens

source_name: PC3 30' Time Point Emetine Treated

source_name: PC3 30' Time Point Un-Treated


title: A404 SMC Differentiation RA96 Replicate 3

status: Public on Jun 30 2004

city: Stanford

first: Joshua

middle: M.

last: Spin

state: CA

address: 300 Pasteur Drive

institute: Stanford University

description:
Time point 3.

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

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

organism: Mus musculus

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

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


title: PC3 2 HrTime Point

description: PC3 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 2 hour time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: PC3 2 Hr Time Point Emetine Treated

source_name: PC3 2 Hr Time Point Un-Treated


title: A404 SMC Differentiation RA96 Replicate 6


title: A404 SMC Differentiation RA48 Replicate 1

description:
Time point 2.

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

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


title: A404 SMC Differentiation Control Replicate 3

description:
Time point 1.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin. Cells were plated in 10-cm dishes and grown to near confluence. Control cells were harvested and RNA was extracted at time 0 with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

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


title: A404 SMC Differentiation RA96 Replicate 5


title: A404 SMC Differentiation RA48 Replicate 5

description:
Time point 2.

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

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


title: A404 SMC Differentiation Control Replicate 1

description:
Time point 1.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin. Cells were plated in 10-cm dishes and grown to near confluence. Control cells were harvested and RNA was extracted at time 0 with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

RNA was purified and isolated using the Qiagen RNeasy Midi Kit Protocol. Ten ug of total RNA were primed with 2 ul of 100uM T16N2 DNA primer at 70 °C for 10 min, and reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase, and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3- or Cy5-dCTP and RNase inhibitor.  RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns. Oligoarray Control Targets and Hybridization Buffer (Agilent In Situ Hybrization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent EZ-Hybe chambers. After hybridization, slides were washed sequentially with 6× SSC/0.005% Triton X-102 and 0.1× SSC/0.005% Triton X-102 before scanning.Slides were hybridized for 17 h at 60°C in a rotating oven, washed, and then scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.6.1.1).
Keywords = smooth muscle cell differentiation
Keywords = A404


title: LNCaP 10' Time Point

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

source_name: LNCaP 10' Time Point Emetine Treated

source_name: LNCaP 10' Time Point Un-Treated


title: A404 SMC Differentiation RA48 Replicate 4


title: A404 SMC Differentiation Control Replicate 5


title: LNCaP 30' Time Point

description: LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 30 min time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: LNCaP 30' Time Point Emetine Treated

source_name: LNCaP 30' Time Point Un-Treated


title: A404 SMC Differentiation Control Replicate 4


title: A404 SMC Differentiation Control Replicate 6


title: A404 SMC Differentiation RA48 Replicate 3


title: PC3 10' Time Point

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

source_name: PC3 10' Time Point Emetine Treated

source_name: PC3 10' Time Point Un-Treated


title: LNCaP 4 HR Time Point

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

source_name: LNCaP 4 HR Time Point Emetine Treated

source_name: LNCaP 4 HR Time Point Un-Treated


title: LNCaP 0' Time Point

description: LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The first time point (0 min) was harvested for both treated and untreated cells. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The Emetine-treatedsample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: LNCaP 0' Time Point Emetine Treated

source_name: LNCaP 0' Time Point Un-Treated


title: A404 SMC Differentiation Control Replicate 2

description:
Time point 1.

Undifferentiated P19 mouse embryonal carcinoma cells, clone A404 obtained from Gary K. Owens at University of Virginia were grown in a-minimum essential medium, 7.5% fetal bovine serum, 200 mg/ml L-glutamine, and penicillin/streptomycin. Cells were plated in 10-cm dishes and grown to near confluence. Control cells were harvested and RNA was extracted at time 0 with Trizol, 5-6 plates per replicate. A common reference consisting of RNA derived from whole 17.5-day mouse embryos was utilized.

RNA was purified and isolated using the Qiagen RNeasy Midi Kit Protocol. Ten ug of total RNA were primed with 2 ul of 100uM T16N2 DNA primer at 70 °C for 10 min, and reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase, and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3- or Cy5-dCTP and RNase inhibitor.  RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns. Oligoarray Control Targets and Hybridization Buffer (Agilent In Situ Hybrization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent EZ-Hybe chambers. After hybridization, slides were washed sequentially with 6× SSC/0.005% Triton X-102 and 0.1× SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, washed, and then scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.6.1.1).
Keywords = smooth muscle cell differentiation
Keywords = A404


title: LNCaP 0' Time Point Self vs. Self

description: LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The Emetine-treatedsample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: LNCaP 0' Time Point Ementine Treated


title: PC3 0' Time Point Self vs. Self

description: PC3 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The Emetine-treatedsample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: PC3 0' Time Point Emetine Treated


title: DU 145 8 HR Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 8 hour  time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 8 HR Time Point Emetine Treated

source_name: DU 145 8 HR Time Point Ementine Un-Treated


title: PC3 0' Time Point

description:
0 TIME POINT

LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The first time point (0 min) was harvested for both treated and untreated cells. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The Emetine-treatedsample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: PC3 0' Time Point Un-Treated


title: VC112, Wild-type versus ddm1 Arabidopsis thaliana

status: Public on Jun 12 2004

city: Austin

first: Matthew

middle: Wayne

last: Vaughn

state: TX

address: 10100 Burnet Rd

department: Texas Advanced Computing Center

institute: University of Texas at Austin

description:
Wild-type and ddm1 bolting plants
Total RNA was extracted with Trizol (Invitrogen) from bolting plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy5 (Wild-type) or Cy3 (ddm1) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: VC110+112

organism: Arabidopsis thaliana

source_name: Wild-type poly(A)+ mRNA

source_name: ddm1 poly(A)+ mRNA


title: VC109, Wild-type versus ddm1 Arabidopsis thaliana

description:
Wild-type and ddm1 eight day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from seedlings, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (Wild-type) or Cy5 (ddm1) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: VC109+111


title: VC111, Wild-type versus ddm1 Arabidopsis thaliana

description:
Wild-type and ddm1 eight day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from seedlings, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy5 (Wild-type) or Cy3 (ddm1) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: VC109+111


title: ES vs. EB

status: Public on May 16 2004

city: Menlo Park

first: Gregory

last: Fisk

address: 230 Constitution Dr.

institute: Geron Corp.

description:
Cell culture:
H9 hESCs were grown using feeder-free conditions for 7 days after passage then either RNA was harvested or embryoid bodies were prepared, cultured in suspension for 4 days, plated on gelatin-coated plates, and cultured for an additional 9 days in embryoid body medium.  Detailed protocols for feeder-free culture and embryoid body formation are available at www.geron.com/scprotocols.pdf

RNA preparation:
Cells were then harvested in situ by overlaying with RLT lysis buffer (Qiagen, Valencia, CA).  RNA was prepared using RNeasy Midi kits as described by the manufacture (Qiagen).  To remove contaminants, RNA was precipitated by adding an equal volume of LiCl Precipitation Solution (Ambion, Austin, TX), washed with 70% ethanol and resuspended in water.

Probe preparation:
Probes were prepared from 20ug of total RNA by direct incorporation of Cy3 or Cy5 dCTP (Amersham Biosciences, Piscataway, NJ) into oligo dT primed first strand cDNA using SuperscriptIII reverse transcriptase (Invitrogen), as described by the dye manufacturer.  RNA was removed by alkaline hydrolysis, then neurtralized probes were purified using Qiaquick columns as described by the manufacturer (Qiagen).

Hybridization/Washing:
Purified probes were dried and resuspended MWG Hybridization Buffer C (MWG Biotech) with the addition of  20 ug of sheared salmon sperm DNA.  Slides were hybridized overnight at 42°.  After the coverslips were removed, the slides were rinsed in 2X SSC + 0.1% SDS, washed in 2X SSC + 0.1% SDS for 5 min, 1X SSC for 5 min, 0.2X SSC for 5 min and 0.1X SSC for 5 min.  Slides were then dried by centrifugation.

Scanning:
Arrays were scanned using a GenePix 4000A microarray scanner (Axon Insturments, Fremont, CA) with 100% scan power and PMT voltage set to minimize saturated pixels and approximately equalize signal intensities in the two channels.  Image processing and data extraction were performed using GenePix Pro 3.0.6 (Axon Instruments).

Data Analysis:
Average background signal from negative control spots was subtracted from Cy3 and Cy5 signal intensities.  In cases where background was larger than signal, signal was set to 1.   Log ratios were calculated for each spot and log ratios from replicate spots were averaged.  Data was normalized using the “Lowess” method, and analyzed for differential expression.  Differentially expressed genes were defined as those with log transformed expression ratios more than 1.5 SD from the mean.  This lower threshold was chosen since comparison of the microarray dat to qRT-PCR on the samples used for the microarray experiment indicated that the data was compressed. For example, TDGF1 was up-regulated 100-fold in uES versus EB by qRT-PCR, but only ~5-fold by microarray analysis in the same sample.  Normalization and identification of differentially expressed genes was performed using the MIDAS package (Saeed et al.  (2003) Biotechniques 34(2):374-8).

source_name: Embryoid bodies (H9 hESCs, 4 day EB+9 days plated)

source_name: undifferentiated hESCs


title: DU 145 10' Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 10 min time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 10' Time Point Emetine Treated

source_name: DU 145 10' Time Point Ementine Un-Treated


title: Estradiol_Ctl_3

status: Public on Jun 19 2004

city: San Antonio

first: Phillip

last: Bowman

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 17β-estradiol only for 5 hrs

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


title: ZMR Sage

status: Public on Jun 01 2004

type: SAGE

anchor: NlaIII

city: Urbana

first: Mark

last: Fredricksen

state: IL

address: 1201 W Gregory Dr

department: Plant Biology

institute: University of Illinois

description:
Dark-grown maize seedlings with primary roots 12-20 mm in length were transplanted to high (-0.03 MPa) water potential vermiculite. About 1,000 roots were collected.  Each root was divided into 4 segments (distances are from the junction of the root apex and root cap): segment 1, 0-3 mm plus the root cap; segment 2, 3-7 mm; segment 3, 7-12 mm; segment 4, 12-20 mm.  Details of the conditions and nutrient modifications have been described before (Sharp et al., 1988; Spollen et al, 2000). From each segment, 250 mg of material was used and RNA was isolated form the combined sample containing all four segments. RNA was isolated using the RNeasy Maxi Kit (Qiagen). RNA integrity was verified by denaturing agarose gel electorphoresis and spectrophotometry.
Fifty µg of total RNA was used to generate the SAGE library (I-SAGE Kit; Invitrogen).

organism: Zea mays

source_name: Root tip well watered


title: EV24, Wild-type versus ddm1/+ backcross mRNA

description:
Wild-type and ddm1/+ backcrossed bolting plants
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (Wild-type) or Cy5 (ddm1/+) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV23+24

source_name: ddm1/+ poly(A)+ mRNA


title: VC1110, Wild-type versus ddm1 Arabidopsis thaliana

description:
Wild-type and ddm1 bolting plants
Total RNA was extracted with Trizol (Invitrogen) from bolting plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (Wild-type) or Cy5 (ddm1) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: VC110+112


title: DU 145 2 HR Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 2 hour  time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 2 HR Time Point Emetine Treated

source_name: DU 145 2 HR Time Point Ementine Un-Treated


title: DU 145 30' Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 30 min time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 30' Time Point Emetine Treated

source_name: DU 145 30' Time Point Ementine Un-Treated


title: EV40, Wild-type versus ddm1 seedlings

description:
Wild-type and ddm1 9 day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy5 (ddm1) or Cy3 (wild-type) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 18 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV39+40


title: EV34, Wild-type versus ddm1 mRNA

description:
Wild-type and ddm1 9 day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (ddm1) or Cy5 (wild-type) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 18 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV33+34


title: EV33, Wild-type versus ddm1 mRNA

description:
Wild-type and ddm1 9 day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy5 (ddm1) or Cy3 (wild-type) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 18 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV33+34


title: EV23, Wild-type versus ddm1/+ backcross mRNA

description:
Wild-type and ddm1/+ backcrossed bolting plants
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy5 (Wild-type) or Cy3 (ddm1/+) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV23+24


title: Estradiol_Ctl_2


title: DU 145 0' Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Simultaneously, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the remaining treated and untreated cells to stop new transcription.  Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 0' Time Point Emetine Treated

source_name: DU 145 0' Time Point Ementine Un-Treated


title: DU 145 0' Time Point Self vs. Self

description: DU 145prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, the first time point (0 min) was harvested for both treated and untreated cells. Cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The Emetine-treatedsample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 0' Time Point Ementine Treated


title: DU 145 1 HR Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 1 hour  time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 1 HR Time Point Emetine Treated

source_name: DU 145 1 HR Time Point Ementine Un-Treated


title: Estradiol_Ctl_4


title: EV39, Wild-type versus ddm1 seedlings

description:
Wild-type and ddm1 9 day-old seedlings
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal).  For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (ddm1) or Cy5 (wild-type) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 18 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV39+40

source_name: Wild-type poly(A)+ mRNA

source_name: ddm1 poly(A)+ mRNA


title: Unfed males - 3A

status: Public on Apr 21 2004

city: Stillwater

first: Richard

middle: C.

last: Essenberg

state: OK

address: 246 NRC

department: Department of Biochemistry and Molecular Biology

institute: Oklahoma State University

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of unfed males using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimaging plate and then scanned with a GS-525 molecular imager (BioRad, Hercules, CA).

organism: Dermacentor andersoni

source_name: Unfed male salivary glands


title: Fed males without females - 3A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimaging plate and then scanned with a GS-525 molecular imager (BioRad, Hercules, CA).

source_name: Fed (without females) male salivary glands


title: Fed males without females - 3

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimager plate and then scanned into a GS-525 molecular imager system (BioRad, Hercules, CA).


title: Fed males with females - 3

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the presence of females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimager plate and then scanned into a GS-525 molecular imager system (BioRad, Hercules, CA).

source_name: Fed (with females) male salivary glands


title: Unfed males - 3

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of unfed males using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimager plate and then scanned into a GS-525 molecular imager system (BioRad, Hercules, CA).


title: Fed males with females - 3A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the presence of females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI phosphorimaging plate and then scanned with a GS-525 molecular imager (BioRad, Hercules, CA).


title: Fed males without females - 2

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to X-ray film for 1 min or 5 min and then scanned into a GS-700 imaging densitometer (BioRad, Hercules, CA).


title: Fed males with females - 2A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the presence of females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to X-ray film for 1 min  or 5 min and then scanned into a GS-700 imaging densitometer (BioRad, Hercules, CA).


title: Unfed males - 2

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of unfed males using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to X-ray film for 1 min or 5 min and then scanned into a GS-700 imaging densitometer (BioRad, Hercules, CA).


title: Fed males without females - 2A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to X-ray film for 1 min or 5 min and then scanned into a GS-700 imaging densitometer (BioRad, Hercules, CA).


title: Fed males with females - 1A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed with females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: Fed males without females - 1A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: Unfed males - 1A

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of unfed males using MMLV reverse transcriptase, then amplified using primers A1 + A3 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: DU 145 4 HR Time Point

description: DU 145 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland).  Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation,  Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 4 hour  time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000).   Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997).  The data were quality filtered with ratio quality method (Chen et al, 2002),  which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: DU 145 4 HR Time Point Emetine Treated

source_name: DU 145 4 HR Time Point Ementine Un-Treated


title: Fed males with females - 2

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the presence of females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP. The membranes were then hybridized in PerfectHyb Plus (Sigma) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA). Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to X-ray film for 1 min or 5 min and then scanned into a GS-700 imaging densitometer (BioRad, Hercules, CA).


title: Unfed males - 1

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of unfed males using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: Fed males without females - 1

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed in the absence of females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: IBD_Colitis Ileum (HG-U133A)

status: Public on Apr 20 2004

city: Regensburg

first: Thomas

last: Langmann

address: Franz-Josef-Strauss-Allee 11

department: University of Regensburg

country: Germany

institute: Institute of Human Genetics

description:
Samples for microarray analysis were derived from terminal ileum and colonic tissues from probands with Crohn´s disease and Ulcerative Colitis and control patients, respectively. All diagnoses of IBD patients were based on classical clinical features, radiological, endoscopic and laboratory findings. IBD tissue biopsies from non-inflamed regions 10 cm distant from pathological areas were selected and sampled by a specialized gastroenterologist. To preserve the transcriptional profile of tissue specimens, biopsies were immediately stabilized in RNAlater solution and kept at –80°C until isolation of RNA. Total RNA was extracted from the tissue biopsies according to the manufacturer´s instructions using the RNeasy Protect Midi Kit (Qiagen). To minimize inter-individual differences in gene expression and to enrich for IBD-specific transcriptional events, 2.5 µg of total RNA from terminal ileum and colon transversum from four individuals of each patient and control group were used for pooling to generate cRNA. Gene expression profiles were determined using Affymetrix HG-U133A and HG-U133B Gene Chips. Array scanning and expression analysis was performed using Microarray Analysis Suite 5.0 software. Each array was scaled to a target intensity of 100 (scaling to all probesets).
Lot batch =

source_name: mucosal biopsy (pool of 4 different donors)


title: IBD_Crohn Colon (HG-U133A)


title: IBD_Crohn Ileum (HG-U133A)


title: IBD_Control Colon (HG-U133B)


title: IBD_Crohn Colon (HG-U133B)


title: IBD_Colitis Colon (HG-U133B)


title: PC3 1 HrTime Point

description: PC3 prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 1 hour time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: PC3 1 Hr Time Point Emetine Treated

source_name: PC3 1 Hr Time Point Un-Treated


title: IBD_Control Ileum (HG-U133B)


title: IBD_Colitis Colon (HG-U133A)


title: poly IC group 1

status: Public on Apr 15 2004

city: Shreveport

first: Stephen

middle: B.

last: Pruett

state: LA

address: 1501 Kings Hwy.

department: Dept. Cellular Biology & Anatomy

institute: LSU Health Sciences Center

description: This is a sample pooled from group one of poly IC-treated mice for an experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Mice were female C57Bl/6 x C3H F1, 12 weeks old.  Mircroarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).  Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice.  Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm).  The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C.  Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C.  After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm.  Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix).  Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms.  Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments.  The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.

source_name: mouse peritoneal macrophages


title: IBD_Crohn Ileum (HG-U133B)


title: IBD_Colitis Ileum (HG-U133B)


title: poly IC group 2

description: This is a sample pooled from group two of poly IC-treated mice (0.1 mg/mouse) for an experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Mice were female C57Bl/6 x C3H F1, 12 weeks old.  Mircroarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).  Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice.  Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm).  The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C.  Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C.  After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm.  Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix).  Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms.  Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments.  The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.


title: Naive control

description: This is the untreated control for an experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Mircroarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).  Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice.  Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm).  The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C.  Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C.  After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm.  Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix).  Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms.  Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments.  The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.


title: Fed males with females - 1

status: Public on Apr 08 2004

description: Complementary DNA’s were synthesized from total RNA obtained from the salivary glands of males fed with females using MMLV reverse transcriptase, then amplified using primers A1 + A2 in the presence of digoxigenin 11-dUTP.  The membranes were then hybridized in 0.25 M sodium phosphate hybridization buffer (pH 7.2) containing 7% SDS and 1 mM ethylene diamine tetra-acetic acid (EDTA) at 65oC. The hybridized membranes were then washed and visualized using CPD-star following a protocol from Tropix (Applied Biosystems, Bedford, MA).  Antidigoxigenin Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN), diluted 1: 10,000 in blocking buffer, were used to detect the digoxigenin-labelled probe. The membranes were exposed to a CI-phosphorimaging plate and then scanned into a phosphorimager GS-700 (BioRad, Hercules, CA).


title: Female-Male(cy3-cy5) #1-5K

status: Public on Mar 27 2004

city: Boston

first: David

middle: J.

last: Waxman

state: MA

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

description:
The genetic expression profile for one male and one female rat liver sample is given as a lowess normalized ratio (f=0.2).  This result is from a single random pairing of one of four males and one of four females.  The purpose of this pairwise comparison is to highlight the sex-dependent differences in rat liver gene expression.
Technical Aspects:  Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were treated with rat growth hormone (GH) (NIDDK, rGH-B-14-SIAFP) given as a continuous infusion using an Alzet osmotic minipump delivering 2 ug GH/100g body weight/h for 7 days.  Intact male and female rats were killed and their livers were collected, frozen in liquid N2 and stored at –80ºC.  Total liver RNA was isolated from frozen liver samples using Trizol reagent (Invitrogen).  Poly(A) RNA was then isolated using  a commercial kit (Ambion).  RNA samples having an A260/A280 ratio >= 1.8 were deemed suitable for use in subsequent analyses.  Total liver RNA and poly(A) RNA samples were treated with DNase I (Ambion).  Poly(A) RNA purified from individual livers was labeled with Cy3-dUTP or Cy5-dUTP (PerkinElmer) in a reverse transcription reaction using 1 ug poly(A) RNA and Superscript II (RNase H mutant) enzyme (Invitrogen) at 39oC for 2 hr.  Unincorporated dNTPs were removed using the Qiaquick PCR-purification kit (Qiagen).  Cy5-labeled male liver cDNA was mixed with Cy3-labeled female or Cy3-labeled GH-treated male liver cDNA followed by co-hybridization of the mixed cDNA population to the microarray for 18 hr at 42oC.  Arrays were washed according to the manufacturer’s protocol.  RNA from each of four male, female, and GH-treated male livers was converted to cDNA and labeled in duplicate.  One series of eight hybridizations (four male + female liver cDNA pairings, and four male + male with GH cDNA pairings)  was carried out on MWG Rat Liver arrays.  A second series containing the same eight hybridizations was carried out on MWG Rat 5K arrays.  The fluorescent cDNA on each microarray was detected using a GenePix 4000B array scanner (Axon Instruments).  The data was preprocessed using GenePix Pro 3.0 (Axon Instruments) and analyzed in GeneSpring 6.0 (Silicon Genetics).  Any spot where there was no fluorescence is reported as 0.  The normalized average ratio is given for all duplicates.
Keywords = Growth hormone
Keywords = liver sexual dimorphism
Keywords = cytochrome P450
Keywords = liver gene expression
Keywords = dual channel cDNA microarray

organism: Rattus norvegicus

source_name: frozen adult female rat liver tissue prepared using Trizol

source_name: frozen adult male rat liver tissue prepared using Trizol


title: IBD_Control Ileum (HG-U133A)


title: Gene expression changes induced by Acyclic Retinoid (GPL787)

status: Public on Apr 03 2004

city: Tokyo

first: Motoyuki

last: Otsuka

department: Department of Gastroenterology

country: Japan

institute: University of Tokyo

description: Total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without ACR treatment for 48 hours. Fluorescent cDNA probes (Cy5 and Cy3-labelled) were prepared from a 30 micro g total RNA isolated from HepG2 with ACR treatment and from a 30 micro g control total RNA isolated from HepG2 cells without ACR treatment, respectively. The different fluorescent-labeled probes were mixed and applied to a microarray following incubation at 60 degree overnight under humidified conditions. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA).  The images were analyzed using ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA) according to the manufacturer’s instructions.

source_name: ACR treated HepG2

source_name: control HepG2 (no treated)


title: Hemisphere #2

city: Kodaira

first: Nobuo

last: Funatsu

state: Tokyo

address: 4-1-1 Ogawahigashi

department: Cellular Biology

institute: National Institute of Neuroscience, NCNP

description: Dissection of E16.5 cortex for RNA extraction.   Cortex Hemispheres of E16.5 mice were divided into the medial (R1), dorsal (R2), ventral (R3), lateral (R4), and occipital regions (R5).   Preparation of RNA and Synthesis of cDNA. Poly A+ RNA was obtained through twice column procedures with oligo dT cellulose column (New England Biolabs, Beverly, MA) and was used for synthesis of cDNA. 1ug of polyA+ RNA was converted into double-stranded cDNA by reverse transcription using ReverTra Ace (ToYoBo, Japan) with oligo dT(24) primers containing the T7 RNA polymerase promotor sequence (Amersham Pharmacia Biotech, Japan). The integrity of RNA was confirmed by agarose electrophoresis and RNA contents were analyzed by measuring the optical density at 260 nm.  Target preparation and array processing. The following procedures including preparation of cRNA targets, hybridization, washing, staining, and scanning were performed as described in the Expression Analysis Technical Manual (Affymetrix, CA). Briefly, biotin-labeled antisense cRNA targets were synthesized in in vitro transcription reaction system (IVT) using cDNA as template (ENZO BioArrayTM HighYieldTM RNA Transcription Labeling kit with T7 RNA polymerase, Enzo, NY). The cRNA targets were size-fragmented to 35-200 bp in a buffer containing potassium and magnesium acetate at 94°C for 35 minutes and were used for hybridization as cRNA targets. The hybridization cocktail [containing 10 ug of target cRNA, control oligo BS (Affymetrix), and Eukaryotic Hybridization controls (Affymetrix)] was hybridized with a mouse U74A array at 42°C for 16 hours in GeneChip Hybridization Oven 640. Washing and staining were performed after hybridization under the fluidics station protocol, EuGE-WS2, on GeneChip Fluidics Station 400. Then, we scanned the array twice on HP GeneArrayTM scanner. Experiments were repeated twice with independent dissection.  Data analysis. To quantify the expression level of a transcript, the average difference value (Adv) was calculated from the intensities against the probe cells using the global methods of normalization by Microarray Suite ver. 4.0 (Affymetrix).

source_name: E16.5 mouse cortex


title: poly IC plus ethanol group 2

description: This is a sample pooled from group two of poly IC-treated mice (0.1 mg/mouse) plus ethanol (6 g/kg) for an experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Mice were female C57Bl/6 x C3H F1, 12 weeks old.  Mircroarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).  Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice.  Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm).  The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C.  Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C.  After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm.  Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix).  Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms.  Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments.  The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.


title: poly IC plus ethanol group 1

description: This is a sample pooled from group one of poly IC-treated mice (0.1 mg/mouse) plus ethanol (6 g/kg) for an experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Mice were female C57Bl/6 x C3H F1, 12 weeks old.  Mircroarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).  Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice.  Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm).  The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C.  Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C.  After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm.  Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix).  Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms.  Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments.  The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.


title: IBD_Control Colon (HG-U133A)


title: Hemisphere #1