title: Leaf_GH2

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 under conditions approximating 25*C by day, 20*C by night with a 16hr photoperiod.  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 0.644164, the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, leaf, expanding leaf
Lot batch = 2003262

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


title: Old Control Day 30 #92 C

status: Public on Aug 21 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 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)

molecule: total RNA

organism: Rattus norvegicus

source_name: soleus muscle


title: Leaf_GH1

status: Public on Aug 27 2003

city: Stanford

first: Erin

middle: A

last: Osborne

state: CA

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 under conditions approximating 25*C by day, 20*C by night with a 16hr photoperiod.  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 0.602709, the ratio which will set GapC (ID_REF = 258588_s_at) to 8500.
Keywords = Tissue Type, leaf, expanding leaf
Lot batch = 2003262

organism: Arabidopsis thaliana

source_name: Genotype: Columbia-0, wild-type. Expanding wild type leaves.  Replicate 2 of 3.


title: Old Control Day 30 #93 C


title: Old Control Day 10 #85 C

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 10 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)


title: Young Control Day 30 #45

status: Public on Aug 28 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: Young 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)


title: Old Control Day 30 #91 C


title: Old Control Day 3 #65 C

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 3 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)


title: Old Control Day 10 #81 C


title: Old Control Day 3 #62 C


title: Old Control Day 3 #63 C


title: Young Control Day 30 #45 C

status: Public on Aug 18 2003


title: Old Control Day 3 #64 C


title: Old Control Day 3 #61 C


title: Old Control Day 10 #83 C


title: Young Control Day 30 #41 C


title: Young Control Day 30 #43 C


title: Young Control Day 30 #42 C


title: Young Control Day 10 #31 C

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: Young Day 10 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)


title: Young Control Day 10 #34 C


title: Young Control Day 6 #24 C

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: Young Day 6 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)


title: Young Control Day 10 #33 C


title: Young Control Day 10 #35 C


title: Old Control Day 10 #84 C


title: Young Control Day 6 #23 C


title: Young Control Day 3 #15 C

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: Young Day 3 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)


title: Young Control Day 3 #12 C


title: Young Control Day 3 #13 C


title: Young Control Day 6 #22 C


title: Young Control Day 3 #11 C


title: Young Control Day 10 #32 C


title: Young Control Day 0 #5 C

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 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 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 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


title: Old Control Day 30 #94


title: Young Control Day 3 #14 C


title: Old Control Day 30 #93


title: Young Control Day 0 #3 C


title: Young Control Day 6 #21 C


title: Old Control Day 30 #92


title: Old Control Day 30 #95


title: Old Control Day 10 #84


title: Young Control Day 0 #4 C


title: Old Control Day 10 #85


title: Old Control Day 10 #83


title: Young Control Day 0 #2 C


title: Old Control Day 30 #91


title: Young Control Day 30 #44 C


title: 14C-II

status: Public on Sep 30 2003

city: Davis

first: Sharon

middle: A.

last: Boylan

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.
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

organism: Mus musculus

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


title: Old Control Day 10 #81


title: Old Control Day 30 #94 B


title: Old Control Day 30 #95 B


title: Old Control Day 10 #85 B


title: Old Control Day 30 #92 B


title: Old Control Day 30 #93 B


title: Old Control Day 3 #64


title: Old Control Day 3 #63


title: mouse_testis_adult_GPL81

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:
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 2.355).
Keywords = Mus musculus, testis, spermatogenesis

source_name: adult testis


title: Old Control Day 10 #82


title: Old Control Day 3 #65


title: Old Control Day 10 #83 B


title: Young Control Day 0 #1 C


title: Old Control Day 10 #84 B


title: Old Control Day 30 #91 B


title: Old Control Day 10 #82 B


title: Old Control Day 3 #62 B


title: Old Control Day 3 #62


title: Old Control Day 3 #61

source_name: soleus


title: Young Control Day 10 #34


title: Old Control Day 3 #65 B


title: Old Control Day 3 #64 B


title: Old Control Day 10 #81 B


title: Young Control Day 10 #35


title: Young Control Day 30 #43 B


title: Young Control Day 30 #45 B


title: Young Control Day 10 #33


title: Young Control Day 10 #32


title: Young Control Day 6 #25


title: Young Control Day 30 #43


title: Young Control Day 30 #42


title: Old Control Day 3 #63 B


title: Young Control Day 30 #41 B


title: Young Control Day 30 #41


title: Young Control Day 30 #42 B


title: Old Control Day 3 #61 B


title: Young Control Day 30 #44 B


title: Young Control Day 10 #35 B


title: Young Control Day 10 #34 B


title: Young Control Day 6 #24


title: Young Control Day 10 #31


title: Young Control Day 6 #23


title: Young Control Day 10 #32 B


title: Young Control Day 10 #31 B


title: Young Control Day 6 #23 B


title: Young Control Day 3 #14


title: Young Control Day 3 #15


title: Young Control Day 6 #21 B


title: Young Control Day 3 #12


title: Young Control Day 6 #22 B


title: Young Control Day 10 #33 B


title: Young Control Day 3 #13 B


title: Young Control Day 3 #11


title: Young Control Day 3 #13


title: Young Control Day 6 #25 B


title: Young Control Day 6 #21


title: Young Control Day 0 #5


title: Young Control Day 6 #22


title: Young Control Day 3 #11 B


title: Young Control Day 0 #4


title: Young Control Day 6 #24 B


title: Young Control Day 3 #12 B


title: Young Control Day 0 #5 B


title: Young Control Day 0 #1


title: Young Control Day 0 #3


title: Young Control Day 0 #2


title: Young Control Day 3 #14 B


title: Young Control Day 0 #1 B


title: Young Control Day 0 #2 B


title: Old Control Day 0 #51

status: Public on Aug 17 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 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
·
*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 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

source_name: soleus skeletal muscle


title: ft-2_7_2

status: Public on Aug 06 2003

city: Tübingen

first: Markus

last: Schmid

address: Speemannstrasse 37-39

department: Molecular Biology

country: Germany

institute: Max Planck Institute for Developmental Biology

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 2
Keywords = flowering

source_name: shoot apex


title: ft-2_7_1

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 1
Keywords = flowering


title: ft-2_5_1

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 1
Keywords = flowering


title: Young Control Day 0 #3 B


title: Young Control Day 0 #4 B


title: ft-2_3_1

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 1
Keywords = flowering


title: ft-2_5_2

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 2
Keywords = flowering


title: Old Control Day 10 #82 C


title: co-2_0_2

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 2
Keywords = flowering


title: co-2_3_1

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 1
Keywords = flowering


title: co-2_5_2

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 2
Keywords = flowering


title: ft-2_0_2

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 2
Keywords = flowering


title: co-2_3_2

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 2
Keywords = flowering


title: ft-2_0_1

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 1
Keywords = flowering


title: co-2_7_2

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 2
Keywords = flowering


title: Ler_7_2

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 2
Keywords = flowering


title: co-2_7_1

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 1
Keywords = flowering


title: Ler_5_2

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 2
Keywords = flowering


title: ft-2_3_2

description:
FT mutant (ft-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 2
Keywords = flowering


title: Ler_7_1

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 1
Keywords = flowering


title: Ler_3_2

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 2
Keywords = flowering


title: co-2_0_1

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 1
Keywords = flowering


title: co-2_5_1

description:
CONSTANS mutant (co-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 1
Keywords = flowering


title: Ler_3_1

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 1
Keywords = flowering


title: Ler_5_1

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 1
Keywords = flowering


title: Ler_0_1

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 1
Keywords = flowering


title: FRI(Sf-2)-flc-3_2

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) and a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 2 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: FRI(Sf-2)-flc-3_0

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) and a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 0 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: lfy-12_5_2

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 2
Keywords = flowering


title: FRI(Sf-2)-flc-3_4

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) and a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 4 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: FRI(Sf-2)-FLC(Col)_4

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 4 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: lfy-12_0_2

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 2
Keywords = flowering


title: lfy-12_7_2

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 2
Keywords = flowering


title: fri(Col)-flc-3_4

description:
Columbia wild type (Col-0) with a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 4 days

Alleles:
FRIGIDA (FRI):                    Col-0
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: lfy-12_0_1

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 1
Keywords = flowering


title: lfy-12_7_1

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 1
Keywords = flowering


title: FRI(Sf-2)-FLC(Col)_2

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 2 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: Young Control Day 30 #44

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: Young 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)


title: Col_7_2

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 2
Keywords = flowering


title: Ler_0_2

description:
Landsberg erecta (Ler) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 0 days
Replicate 2
Keywords = flowering


title: FRI(Sf-2)-FLC(Col)_0

description:
Columbia wild type (Col-0) with an active FRI allele introgressed from San Feliu-2 (Sf-2) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 0 days

Alleles:
FRIGIDA (FRI):            Sf-2
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: Col_7_1

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 7 days
Replicate 1
Keywords = flowering


title: fri(Col)-flc-3_0

description:
Columbia wild type (Col-0) with a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 0 days

Alleles:
FRIGIDA (FRI):            Col-0
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: Col_5_2

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 2
Keywords = flowering


title: fri(Col)-flc-3_2

description:
Columbia wild type (Col-0) with a deletion in FLC grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 2 days

Alleles:
FRIGIDA (FRI):                    Col-0
FLOWERING LOCUS C (FLC):  flc-3
Keywords = flowering


title: lfy-12_3_2

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 2
Keywords = flowering


title: Col_5_1

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 1
Keywords = flowering


title: fri(Col)-FLC(Col)_4

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 4 days

Alleles:
FRIGIDA (FRI):                    Col-0
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: lfy-12_3_1

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 1
Keywords = flowering


title: Col_3_2

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 2
Keywords = flowering


title: lfy-12_5_1

description:
LEAFY mutant (lfy-12) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 5 days
Replicate 1
Keywords = flowering


title: fri(Col)-FLC(Col)_0

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 0 days

Alleles:
FRIGIDA (FRI):                    Col-0
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: CD8+ E/EM T cells (SubC)

status: Public on Jul 25 2003

city: Denver

first: Brad

last: Swanson

state: CO

institute: National Jewish Medical and Research Center/HHMI

description:
•In vitro generated CD8+ effector/effector memory (CD8IL-2) T cells were generated from female P14 T cell receptor transgenic mice on the C57Bl/6 genetic background.

•Whole spleen and lymph node cells from female P14 T cell receptor transgenic mice were homogenized and stimulated with cognate peptide (KAVYNFATM) for 2 days.  The cells were then grown in IL-2 (effector/effector memory/CD8IL-2 ) for 7 additional days.  Effector/effector memory (CD8IL-2) CD8+ T cells were then sorted by high speed cell sorting for the predominant CD62Llow population of cells.

source_name: in vitro generated CD8+ effector/effector memory T cells


title: CD8+ CM T cells (SubC)

description:
•In vitro generated CD8+ central memory(CD8IL-15) T cells were generated from female P14 T cell receptor transgenic mice on the C57Bl/6 genetic background.

•Whole spleen and lymph node cells from female P14 T cell receptor transgenic mice were homogenized and stimulated with cognate peptide (KAVYNFATM) for 2 days.  The cells were then grown in IL-15 (central memory/CD8IL-15) for 7 additional days.  Central memory (CD8IL-15) CD8+ T cells were sorted by high speed cell sorting for the predominant CD62Lhigh population of cells.

source_name: in vitro generated CD8+ central memory T cells


title: CD8+ E/EM T cells (SubB)


title: Col_3_1

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point 3 days
Replicate 1
Keywords = flowering


title: fri(Col)-FLC(Col)_2

description:
Columbia wild type (Col-0) grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D).

Time point: 2 days

Alleles:
FRIGIDA (FRI):            Col-0
FLOWERING LOCUS C (FLC):  Col-0
Keywords = flowering


title: CD8+ CM T cells (SubB)

description:
•In vitro generated CD8+ central memory(CD8IL-15) T cells were generated from female P14 T cell receptor transgenic mice on the C57Bl/6 genetic background.

•Whole spleen and lymph node cells from female P14 T cell receptor transgenic mice were homogenized and stimulated with cognate peptide (KAVYNFATM) for 2 days.  The cells were then grown in IL-15 (central memory/CD8IL-15) for 7 additional days.  Central memory (CD8IL-15) CD8+ T cells were sorted by high speed cell sorting for the predominant CD62Lhigh population of cells.
Keywords = CD8, T cells, central memory, effector, effector memory