title: Old Treatment Day 0 #60 A

description:
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 2:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 2-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	 Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)


title: Old Treatment Day 0 #57 B

status: Public on Oct 05 2003

type: RNA

city: Columbia

first: Scott

last: Pattison

state: MO

country: USA

institute: University of Missouri

description:
II.	Experiment Description
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 2:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 2-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	 Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)

molecule: total RNA

organism: Rattus norvegicus

source_name: soleus muscle


title: Old Treatment Day 0 #60 B


title: Old Treatment Day 10 #86 A

description:
II.	Experiment Description
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 10 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)


title: Young Treatment Day 0 #9 B

description:
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Young Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 2:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 2-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	 Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)


title: Young Treatment Day 0 #10 A


title: Old Treatment Day 0 #56 B


title: Young Treatment Day 0 #8 B


title: Old Treatment Day 0 #57 A


title: Young Treatment Day 0 #10 B


title: Old Treatment Day 0 #59 B


title: Old Treatment Day 0 #58 A


title: Old Treatment Day 0 #59 A


title: Young Treatment Day 0 #8 A

source_name: soleus skeletal muscle


title: Young Treatment Day 0 #9 A


title: Old Treatment Day 0 #56 A


title: Young Treatment Day 0 #6 B


title: Young Treatment Day 0 #7 B


title: Young Treatment Day 0 #7

description:
II.	Experiment Description
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Young Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 2:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 2-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	 Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)


title: mouse_testis_interstitial_cells_GPL83

status: Public on Oct 02 2003

city: New York

first: Nikolaus

last: Schultz

state: NY

address: 1275 York Ave, Box 460

department: Computational Biology Center

institute: Memorial Sloan-Kettering Cancer Center

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 5.232).
Keywords = Mus musculus, testis spermatogenesis

organism: Mus musculus

source_name: 19 day old testis, interstitial cells


title: Young Treatment Day 0 #6 A


title: mouse_testis_day_26_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.098).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 26 day old testis


title: mouse_testis_day_14_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.223).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 14 day old testis


title: mouse_testis_day_11_replicate_2_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.401).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 11 day old testis


title: mouse_testis_day_29_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 4.168).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 29 day old testis


title: mouse_testis_day_18_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 4.965).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 18 day old testis


title: mouse_testis_day_21_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.953).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 21 day old testis


title: mouse_testis_day_08_replicate_2_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 8.788).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 8 day old testis


title: mouse_testis_day_08_replicate_1_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 4.814).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_11_replicate_1_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 5.578).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_1_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 7.191).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 1 day old testis


title: mouse_testis_day_21_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.228).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_2_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 5.502).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 4 day old testis


title: mouse_testis_day_18_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.022).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_interstitial_cells_GPL82

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.068).
Keywords = Mus musculus, testis spermatogenesis


title: mouse_testis_day_26_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.978).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_08_replicate_1_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.669).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_3_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 6.247).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_11_replicate_2_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.984).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_2_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.771).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_1_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 5.182).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_1_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 7.485).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_11_replicate_1_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.200).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_08_replicate_2_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.865).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_29_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.647).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_adult_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.616).
Keywords = Mus musculus, testis, spermatogenesis

source_name: adult testis


title: mouse_testis_day_14_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.948).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_1_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.821).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_26_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.960).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_21_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.930).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_3_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.978).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_interstitial_cells_GPL81

description:
RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. 50 ng total RNA were amplified using two rounds of amplification. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.265).
Keywords = Mus musculus, testis spermatogenesis


title: mouse_testis_day_01_replicate_2_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 3.891).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_adult_GPL82

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 2.093).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_29_GPL81


title: mouse_testis_day_08_replicate_2_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.042).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_08_replicate_1_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.569).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_18_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.959).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_2_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.290).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_11_replicate_1_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.293).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_3_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.140).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_01_replicate_1_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.192).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_2_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 1.029).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_14_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.348).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_04_replicate_1_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.880).
Keywords = Mus musculus, testis, spermatogenesis


title: mouse_testis_day_11_replicate_2_GPL81

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 0.364).
Keywords = Mus musculus, testis, spermatogenesis


title: 14C-I

status: Public on Sep 30 2003

city: Davis

first: Sharon

middle: A.

last: Boylan

state: CA

address: 2403 Tupper Hall

department: Biological Chemistry

institute: University of California, Davis

description:
I. Exp Design
1. Type of experiment: Young vs Aged comparison
2. Experimental factors: 2 month old mouse vs 24 month old mouse
3. How many hybridizations in exp: 12
4. If a common reference used for all the hybs: No
5. Quality Control steps: three independent replicates for each age
6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye

II. Samples Used, extract prep, and labeling
1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging
Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye
2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina.  The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 microliter of RNAlater buffer (QIAgen, Valencia, CA).
3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA.
4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification.
5. No external controls were added.

III. Hybridization procedures and parameters
1. Sample , array type, batch and serial # used
2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution.  Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm.  30 minute washes at 65ºC with 2XSSC, 1%SDS (3X); 0.5XSSC, 0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).

IV. Measurement data and specifications of data processing
1,2.  Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50 micron resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis.
Keywords = retinal pigmented epithelium
Keywords = aging
Keywords = choroid
Lot batch = 1040052

source_name: retinal pigmented epithelium/choroid from 24 month old male


title: 12C-II

description:
I. Exp Design
1. Type of experiment: Young vs Aged comparison
2. Experimental factors: 2 month old mouse vs 24 month old mouse
3. How many hybridizations in exp: 12
4. If a common reference used for all the hybs: No
5. Quality Control steps: three independent replicates for each age
6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye

II. Samples Used, extract prep, and labeling
1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging
Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye
2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina.  The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 microliter of RNAlater buffer (QIAgen, Valencia, CA).
3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA.
4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification.
5. No external controls were added.

III. Hybridization procedures and parameters
1. Sample , array type, batch and serial # used
2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution.  Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm.  30 minute washes at 65ºC with 2XSSC, 1%SDS (3X); 0.5XSSC, 0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).

IV. Measurement data and specifications of data processing
1,2.  Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50 micron resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis.
3.  Data processing:  A gene was identified as expressed if its background subtracted intensity was greater than 1.4 times background.  The data were normalized using a simple global scaling procedure, and the Significance Analysis of Microarrays (SAM, version 1.15) program was used to determine significant differential gene expression.
Keywords = retinal pigmented epithelium, aging, choroid
Lot batch = 0060486


title: 13C-II


title: 13C-I


title: 5C-II

description:
I. Exp Design
1. Type of experiment: Young vs Aged comparison
2. Experimental factors: 2 month old mouse vs 24 month old mouse
3. How many hybridizations in exp: 12
4. If a common reference used for all the hybs: No
5. Quality Control steps: three independent replicates for each age
6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye

II. Samples Used, extract prep, and labeling
1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging
Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye
2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina.  The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 microliter of RNAlater buffer (QIAgen, Valencia, CA).
3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA.
4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification.
5. No external controls were added.

III. Hybridization procedures and parameters
1. Sample , array type, batch and serial # used
2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution.  Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm.  30 minute washes at 65ºC with 2XSSC, 1%SDS (3X); 0.5XSSC, 0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).

IV. Measurement data and specifications of data processing
1,2.  Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50 micron resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis.
Keywords = retinal pigmented epithelium
Keywords = aging
Keywords = choroid
Lot batch = 0060486

source_name: retinal pigmented epithelium/choroid from 2 month old male


title: 12C-I


title: 1C-II


title: 3C-I

description:
I. Exp Design
1. Type of experiment: Young vs Aged comparison
2. Experimental factors: 2 month old mouse vs 24 month old mouse
3. How many hybridizations in exp: 12
4. If a common reference used for all the hybs: No
5. Quality Control steps: three independent replicates for each age
6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye

II. Samples Used, extract prep, and labeling
1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging
Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye
2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina.  The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 microliter of RNAlater buffer (QIAgen, Valencia, CA).
3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA.
4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification.
5. No external controls were added.

III. Hybridization procedures and parameters
1. Sample , array type, batch and serial # used
2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution.  Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm.  30 minute washes at 65ºC with 2XSSC, 1%SDS (3X); 0.5XSSC, 0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).

IV. Measurement data and specifications of data processing
1,2.  Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50 micron resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis.
3.  Data processing:  A gene was identified as expressed if its background subtracted intensity was greater than 1.4 times background.  The data were normalized using a simple global scaling procedure, and the Significance Analysis of Microarrays (SAM, version 1.15) program was used to determine significant differential gene expression.
Keywords = retinal pigmented epithelium, aging, choroid
Lot batch = 1040052


title: Effect of iron chelation (SIH) on human cytomegalovirus infected MRC-5 fibroblasts (30 hours PE)

city: Syracuse, New York

first: Lilia

last: Maglova-Crowe

address: 130 College Place, BRL

department: Biology

institute: Syracuse University

description:
MRC-5 cells (American Type Culture Collection, Manassas, VA) derived from human lung fibroblasts (passages 19-25) were cultured in MEM with Earle’s salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were grown in an incubator with a humidified atmosphere of 5% CO2 in air at 37oC for several days to reach confluence.
HCMV strain AD169 (American Type Culture Collection, Manassas, VA) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells. At the appropriate time, the infected cells were lysed and the infectivity of the resultant viral stock was assayed by plaque assay. For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection (MOI) of ~3-5 plaque-forming units (PFU) per cell for 1 h at 370C. To control for non-specific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells were always mock infected by a 1h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with phosphate buffered saline (PBS) followed by fresh culture medium and returned to cell culture for various post-exposure (PE) times.
Mock- or HCMV-infected cells were homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA following the method of Chirgwin et al. (PubMed ID 518835). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Two successive oligo (dT) cellulose columns were then used to isolate poly A+ RNA for microarray analysis, yielding 600 ng of mRNA per channel (mock-infected or HCMV-infected cells). These were labeled with Cy3 and Cy5 dyes respectively prior to hybridization with a UniGEM V cDNA microarray (Incyte Genomics, St Louis, MO)
Keywords = human cytomegalovirus
Keywords = MRC-5 fibroblasts
Keywords = iron chelator
Keywords = salicylaldehyde isonicotinoyl hydrazone

organism: Homo sapiens

source_name: HCMV-infected MRC-5 fibroblasts + iron chelator (SIH, 100 microM)

source_name: HCMV-infected MRC-5 fibroblasts


title: 5C-I


title: 3C-II


title: 1C-I

description:
I. Exp Design
1. Type of experiment: Young vs Aged comparison
2. Experimental factors: 2 month old mouse vs 24 month old mouse
3. How many hybridizations in exp: 12
4. If a common reference used for all the hybs: No
5. Quality Control steps: three independent replicates for each age
6. Description – Young mice were compared to old mice to determine gene changes that occur with aging in the mouse retinal pigmented epithelium/choroid of the eye

II. Samples Used, extract prep, and labeling
1. Biosource: Mus musculus strain C57BL/6 from National Institute on Aging
Old mice available from National Institute on Aging, young mice from Jackson Labs
Male mice, 2 and 24 months old, used only a portion of the eye, the retinal pigmented epithelium in combination with the choroid (RPE/choroid) with no tissue from other parts of the eye
2. Manipulations: Animals were allowed to acclimate to our facilities for two weeks prior to death by cervical dislocation- no treatments were performed. Eyes were removed from each mouse, the anterior segments were removed with a circumferential incision, and posterior segments were then separated from the whole retina.  The pigmented layer including RPE/choroid was scraped gently from the sclera as a sheet in less than 50 ml of RNAlater buffer (QIAgen, Valencia, CA).
3. Extract preparation: RPE/choroid tissue was placed into lysis buffer RLT (Qiagen) and homogenized using needle and syringe followed by QIAshredder (Qiagen). Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer’s instructions. DNase treatment (Qiagen) was used to remove any DNA contaminants. The Atlas SMART PCR system from Clontech was used to amplify the RNA using PCR following the manufacturer’s instructions starting from 500 ng total RNA.
4. Labeling protocol: the SMART cDNA probe labeling kit from Clontech was used to prepare 33P-labeled cDNA starting with 500 ng of purified cDNA resulting from amplification.
5. No external controls were added.

III. Hybridization procedures and parameters
1. Sample , array type, batch and serial # used
2. Hybridization protocol: Each hybridization reaction contained 500 ng labeled cDNA (denatured at 100ºC) in 10 ml ExpressHyb (for a single array). Arrays were blocked with SMART Blocking solution.  Hybridization was done in 10 ml volume at 65ºC overnight using a hybridization oven set at 5-7rpm.  Wash at 65ºC with 2XSSC,1%SDS (3X); 0.5XSSC,0.5%SDS (1X); and 0.33XSSC,0.5%SDS (1X).

IV. Measurement data and specifications of data processing
1,2.  Arrays were exposed to a phosphorimaging screen for 3-5 days and scanned at 50mm resolution using the Storm 860 (Molecular Dynamics, Sunnyvale, CA). Gel images from the phosphorimager were exported to the AtlasImage 1.5 software (Clontech) for analysis.
3.  Data processing:  A gene was identified as expressed if its background subtracted intensity was greater than 1.4 times background.  The data were normalized using a simple global scaling procedure, and the Significance Analysis of Microarrays (SAM, version 1.15) program was used to determine significant differential gene expression.
Keywords = retinal pigmented epithelium, aging, choroid


title: Human cytomegalovirus vs mock-infected MRC-5 fibroblasts (72 hours PE)

status: Public on Sep 20 2003

description:
MRC-5 cells (American Type Culture Collection, Manassas, VA) derived from human lung fibroblasts (passages 19-25) were cultured in MEM with Earle’s salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were grown in an incubator with a humidified atmosphere of 5% CO2 in air at 37oC for several days to reach confluence.
HCMV strain AD169 (American Type Culture Collection, Manassas, VA) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells.  At the appropriate time, the infected cells were lysed and the infectivity of the resultant viral stock was assayed by plaque assay. For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection (MOI) of ~3-5 plaque-forming units (PFU) per cell for 1 h at 370C.  To control for non-specific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells were always mock infected by a 1h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with phosphate buffered saline (PBS) followed by fresh culture medium and returned to cell culture for various post-exposure (PE) times.
Mock- or HCMV-infected cells were homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA following the method of Chirgwin et al. (PubMed ID 518835). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Two successive oligo (dT) cellulose columns were then used to isolate poly A+ RNA for microarray analysis, yielding 600 ng of mRNA per channel (mock-infected or HCMV-infected cells). These were labeled with Cy3 and Cy5 dyes respectively prior to hybridization with a UniGEM V cDNA microarray (Incyte Genomics, St Louis, MO).
Keywords = human cytomegalovirus
Keywords = MRC-5 fibroblasts

source_name: HCMV-infected MRC-5 fibroblasts (72 hours PE)

source_name: Mock-infected MRC-5 fibroblasts


title: Vitamin K2 to HepG2 cells

status: Public on Sep 29 2003

city: Tokyo

first: Motoyuki

last: Otsuka

department: Department of Gastroenterology

country: Japan

institute: University of Tokyo

description: Human hepatocellular carcinoma cells, HepG2 , cultured in DMEM containing 10% fetal calf serum.To study gene expression changes induced by Vitamin K2, total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without 50 microM of Vitamin K2 treatment for three days. The cDNA microarrays Human Oligo Chip 30K “AceGene” subset A was purchased from Hitachi Software Engineering Co. (Yokohama-City, Japan). This array contains 10368 kinds of gene-specific 50 mer sense oligonucleotides. Subset A contains mainly known function ORF oligo probes. Preparation of fluorescent cDNA targets by an indirect labeling approach was performed using PowerScript reverse transcription kit (Clontech). First-strand cDNAs were synthesized from 30 microg of total RNA from HepG2 cells treated with or without Vitamin K2 using random primer and PoweScript Fluorescent Labelling Kit. Monofunctional, N-hydroxysuccinimide-activated fluorescent dyes (Cy3 and Cy5; Amersham) were coupled to the cDNAs by reaction with the amino functional groups. Untreated cells were labeled with Cy3 as a control and treated cells with Cy5 as a sample. After free dyes were removed using MinElute Purification kit (Qiagen), the targets were mixed together and added to the microarrays, and then incubated overnight (16 hours) at 42?C. The slides were washed at 30?C in each with 2x SSC, 0.1% SDS and 2x SSC for 10 min and 5 min, respectively, and washed in 1x SSC for 5 min. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA). The Cy3 and Cy5 intensities with background subtraction were determined by ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA). The expression data were then filtered based on their channel intensities, spot size and flag (missing or inaccurate data), and the Cy5/Cy3 ratios were calculated and normalized by median-centering the log-ratio of all genes.

source_name: HepG2 cells added by Vitamin K2

source_name: HepG2 cells without Vitamin K2


title: Human cytomegalovirus vs mock-infected MRC-5 fibroblasts (30hours PE)

description:
MRC-5 cells (American Type Culture Collection, Manassas, VA) derived from human lung fibroblasts (passages 19-25) were cultured in MEM with Earle’s salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were grown in an incubator with a humidified atmosphere of 5% CO2 in air at 37oC for several days to reach confluence.   HCMV strain AD169 (American Type Culture Collection, Manassas, VA) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells.  At the appropriate time, the infected cells were lysed and the infectivity of the resultant viral stock was assayed by plaque assay. For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection (MOI) of ~3-5 plaque-forming units (PFU) per cell for 1 h at 370C.  To control for non-specific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells were always mock infected by a 1h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with phosphate buffered saline (PBS) followed by fresh culture medium and returned to cell culture for various post-exposure (PE) times.   Mock- or HCMV-infected cells were homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA following the method of Chirgwin et al. (PubMed ID 518835). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Two successive oligo (dT) cellulose columns were then used to isolate poly A+ RNA for microarray analysis, yielding 600 ng of mRNA per channel (mock-infected or HCMV-infected cells). These were labeled with Cy3 and Cy5 dyes respectively prior to hybridization with a UniGEM V cDNA microarray (Incyte Genomics, St Louis, MO).
Keywords = human cytomegalovirus
Keywords = MRC-5 fibroblasts

source_name: HCMV-infected MRC-5 fibroblasts (30 hours PE)


title: h5

status: Public on Sep 17 2003

city: Columbus

first: Herbert

last: Auer

state: OH

address: 420 West 12th Ave

institute: Human Cancer Genetics

description:
PBMCs were shock-frozen in liquid nitrogen and thawed, after which cells were left at room temperature for 5 minutes before RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol.  Targets were produced using standard Affymetrix procedures from about 8ug of total RNA.
Keywords = Homo sapiens, RNA degradation, microarray

source_name: Peripheral blood mononuclear cells (PBMCs)


title: FLOWER_GH5

status: Public on Aug 27 2003

city: Stanford

first: Erin

middle: A

last: Osborne

address: 260 Panama Dr.

department: Department of Plant Biology

institute: Carnegie Institute of Washington

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a green house approximating 25*C by day, 20*C by night with a 16 hr photoperiod.  Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 0.91663 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, flower
Lot batch = 2003262

organism: Arabidopsis thaliana

source_name: Genotype: Columbia-0, wild-type; Treatment: wild type flowers.  Replicate 3 of 4.


title: n5


title: FLOWER_GH6

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a green house approximating 25*C by day, 20*C by night.  Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 1.345746 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, flower
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: wild type flowers.  Replicate 4 of 4.


title: n1

description:
PBMCs were shock-frozen in liquid nitrogen and thawed, after which cells were left at room temperature for 0 minutes before RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Targets were produced using standard Affymetrix procedures from about 8ug of total RNA.
Keywords = Homo sapiens, RNA degradation, microarray


title: FLOWER_GC5

description:
Growth Conditions and Inoculations:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 0.897192 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, flower
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: wild type flowers.  Replicate 1 of 4.


title: STEM_GC7

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Expanding upper 2" of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 0.971129 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, stem
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: Expanding wild type stems.  Replicate 1 of 4.


title: mouse_testis_day_01_replicate_2_GPL83

description:
Full testis RNA preparation was performed using RNA Stat-60 (Teltest, Friendswood, TX), according to the manufacturer’s protocol. Detection calls were calculated using a Tau value of 0.015. Signal intensities were scaled to an average of 250 (scale factor 6.915).
Keywords = Mus musculus, testis, spermatogenesis

source_name: 1 day old testis


title: STEM_GH7

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a green house approximating 25*C by day, 20*C by night with 16 hr photoperiods.  Expanding upper 2" of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 1.132473 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, stem
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: Expanding wild type stems.  Replicate 3 of 4.


title: STEM_GH8

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Expanding upper 2" of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 0.932386 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, stem
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: Expanding wild type stems.  Replicate 4 of 4.


title: FLOWER_GC6

description:
Growth Conditions and Inoculations:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Developed flowers and unopened buds were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 1.07150 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, flower
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: wild type flowers.  Replicate 2 of 4.


title: STEM_GC8

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Expanding upper 2" of the stem with siliques and pedicels removed were harvested 29 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method (Invitrogen, Ramonell et al. (2002) Mol. Plant Pathol. 3: 301) and purified with a silica membrane column (Qiagen, RNeasy). Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying signal intensities by 1.072704 the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, stem
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type; Treatment: Expanding wild type stems.  Replicate 2 of 4.


title: h1


title: Old Control Day 30 #94 C

status: Public on Aug 21 2003

description:
II.	Experiment Description
1)	Experimental Design
·	Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
·	Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
·	Contact: boothf@missouri.edu
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 1:
·	Experimental Factors: Age
·	How many hybridizations in the experiment: 45 unique samples were hybridized once per array x 3 arrays each = 135 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young control soleus muscles and old control soleus muscles.
·	Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 30 Control muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

Ø	Biomaterial manipulations
·	Growth conditions: normal
controlled environment
20-22 ºC average temperature
housed in guinea pig cages 2-3/cage
rubber mats covered the bottoms of cages
12/12 hour light/dark cycle
regular rat chow was provided ad libitum
water was available ad libitum
½” x 3” poplar dowel rods doused in apple juice were given for environmental enhancement
·	Treatments: N/A
·	Tissue handling: both soleus muscles from a single rat were excised, weighed, and immediately frozen in liquid N2, the muscles were then stored at –80ºC until RNA isolation.

Ø	Extract preparation
·	Extraction method: muscle tissue was powdered over liquid N2 then put into TRIzol solution and homogenized with a Polytron homogenizer for 3 bouts of 15 seconds on setting 7.  Total RNA was extracted as per manufacturer instructions.  Total RNA from each sample was used to prepare biotinylated target RNA, with minor modifications from the manufacturers recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).  Briefly, 10 µg of total RNA was used to generate first-strand cDNA by using a T7-oligo(dT24) primer.  Following, second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics), resulting in approximately 100-fold amplification of RNA.  Finally, the biotinylated populations of cRNA were fragmented according to the linked protocol.  A complete description of procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Minor modifications: All incubations were done in a thermocycler instead of a water bath, cDNA was quantified follwing cDNA synthesis, prior to in vitro transcription, using a PicoGreen kit (Molecular Probes). One microgram of cDNA was then in vitro transcribed.
·	Target Preparation: Target cDNA generated from each sample were then processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Quality control: The quality and amount of starting RNA, cDNA, cRNA, and fragmented cRNA were confirmed using agarose gel analyses.
·	Spike controls:  Briefly, spike controls were added to 10 µg fragmented cRNA before prior to hybridization. A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Hybridization Procedures
·	Hybridization:  Arrays were pre-hybridized, loaded, and hybridized for 16 hours prior to washing and staining.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx
·	Washing and Staining: Arrays were then washed and stained with streptavidin-phycoerythrin, before being scanned on an Affymetrix GeneChip scanner.  A complete description of these procedures is available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

Ø	Measurement data and specifications of data processing
·	Scanning:  The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
·	Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment.  The corners were examined by eye for proper grid alignment.  Images were also scanned for the absence of bubbles, scratches, and speckling.  If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses.  3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0.  From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
·	Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities.  No further normalization was applied.
·	Data Analysis:
·	Experiment 1:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a Student’s t-test.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 (raw p=4.478x10-6) were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
·	Experiment 3:
·	Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed.  The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 3-way ANOVA.  A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
·	The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)

source_name: soleus muscle


title: Leaf_GC2

description:
Growth Conditions:  Columbia (Col-0) Arabidopsis thaliana plants were grown at a density of 4 plants per 5" square pot in a growth chamber set to 25*C by day, 20*C by night.  Days were set to a 16hr photoperiod with 125 micro mol m-2s-1 fluorescent irradiation.  Expanding leaves were harvested 15 days post germination in the middle of the photoperiod.
RNA and Microarray Methods: Total RNA was extracted from the plants using the Trizol method. Twenty micrograms biotinylated complementary RNA (cRNA) was prepared as described (Hernan et al. (2003) Cancer Res. 63, 140) from the purified total RNA. The resulting cRNA was used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix Microarray Suite 5.0 software with the target intensity set to 500. Normalized signal intensities were generated by multiplying raw signal intensities by 0.7974257, the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, leaf,
Lot batch = 2003262

source_name: Genotype: Columbia-0, wild-type. Expanding wild type leaves.  Replicate 1 of 3.


title: Old Control Day 30 #95 C