title: ARPE-19 cells grown on collagen I.2

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.


title: ARPE-19 cells grown on fibronectin.2

status: Public on Oct 15 2003

type: RNA

city: Baltimore

first: James

middle: T

last: Handa

state: MD

address: 600 N. Wolfe St.

department: Wilmer Eye Institute

country: USA

institute: Johns Hopkins Medical Institutes

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Keywords = retinal pigment epithelium
Keywords = ARPE-19
Keywords = macula
Keywords = extracellular matrix

molecule: total RNA

organism: Homo sapiens

source_name: ARPE-19 cells


title: ARPE-19 cells grown on fibronectin.3


title: ARPE-19 cells grown on collagen IV.2


title: ARPE-19 cells grown on collagen IV.1


title: ARPE-19 cells grown on collagen IV.3


title: ARPE-19 cells grown on fibronectin.1

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Keywords = pigment epithelium
Keywords = ARPE-19
Keywords = macula
Keywords = extracellular matrix


title: ARPE-19 grown on collagen I


title: ARPE-19 cells grown on plastic.1


title: 3-aminopicoline-5-carboxylic acid (pichloram) 20 minutes treatment

status: Public on Oct 12 2003

city: RTP,

first: Yang

last: Qiu

state: NC

address: 108 TW Alexander Dr.

department: Investigational Genomics

institute: Paradigm Genetics, Inc

description:
Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.
Keywords = Auxin
Keywords = Pichloram
Keywords = Arabidopsis

organism: Arabidopsis thaliana

source_name: extracted from 20 mins 0.1% ethanol treatment of 4 d old Col-0 seedlings

source_name: extracted from 20 mins 10uM 3-aminopicoline-5-carboxylic acid (pichloram) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: AcB61 #12250573

status: Public on Oct 06 2003

city: Montreal

first: Gundula

last: Min-Oo

state: Quebec

department: Biochemistry

country: Canada

institute: McGill University

description:
Animals were sacrificed and organs were rapidly frozen in liquid nitrogen, and subsequently stored at <ETH>80oC. Tissues were mechanically homogenized with a polytron  and total cellular RNA was extracted using a commercially available  reagent (TMTRIzol; Invitrogen).  The poly-(A)+ fraction of the RNA was isolated by chromatography  with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum.
The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation.
Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings.
Keywords = spleen
Keywords = malaria

organism: Mus musculus

source_name: Spleen of AcB61 mice

source_name: Spleen of A/J mice


title: AcB61 #12249620


title: 3,7 dichloroquinoline-8-carboxylic acid (quinclorac) 20 minutes treatment

description:
Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.
Keywords = Auxin
Keywords = Quinclorac
Keywords = Arabidopsis

source_name: extracted from 20 mins 10uM 3,7 dichloroquinoline-8-carboxylic acid (quinclorac) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: indole-3-acetic acid (IAA) 60 minutes treatment

description:
Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.
Keywords = Auxin
Keywords = IAA
Keywords = Arabidopsis

source_name: extracted from 60 mins 0.1% ethanol treatment of 4 d old Col-0 seedlings

source_name: extracted from 60 mins 10uM indole-3-acetic acid (IAA) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: AcB61 #12250554


title: indole-3-acetic acid (IAA) 40 minutes treatment

source_name: extracted from 40 mins 0.1% ethanol treatment of 4 d old Col-0 seedlings

source_name: extracted from 40 mins 10uM indole-3-acetic acid (IAA) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: 2,4-dichlorophenoxy-1-acetic acid 20 minutes treatment

description: Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.

source_name: extracted from 20 mins 10uM 2,4-dichlorophenoxy-1-acetic acid in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: naphthalene-1- acetic acid (NAA) 20 minutes treatment

description:
Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.
Keywords = Auxin
Keywords = NAA
Keywords = Arabidopsis

source_name: extracted from 20 mins 10uM naphthalene-1- acetic acid (NAA) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: AcB61 #12243339


title: AcB61 #12250566


title: AcB61 #12243360


title: 5-methoxy benzoic acid (dicamba) 20 minutes treatment

description:
Approximately 500 surface sterilized seeds of Arabidopsis seeds (ecotype: Col-0) were sterilely sown on filter disks overlaying solid ½ MS media 1% sucrose, stratified at 4C for 48 h and grown in darkness at 25C for 4 d. Seedlings were gently submerged by application to the plates of the different auxin solutions (made in ethanol carrier; 0.1% final concentration) and at the indicated times, the seedlings were lifted from the plate en mass and flash frozen in liquid nitrogen.
Keywords = Auxin
Keywords = Dicamba
Keywords = Arabidopsis

source_name: extracted from 20 mins 10uM 5-methoxy benzoic acid (dicamba) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: AcB55 #12262226

source_name: Spleen of AcB55 mice


title: ARPE-19 cells grown on Matrigel.2


title: AcB55 #12256790b


title: Old Control Day 6 #75 C

status: Public on Oct 05 2003

city: Columbia

first: Scott

last: Pattison

state: MO

institute: University of Missouri

description:
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 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 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)

organism: Rattus norvegicus

source_name: soleus muscle


title: AcB55 #12262264


title: indole-3-acetic acid (IAA) 20 minutes treatment

source_name: extracted from 20 mins 10uM indole-3-acetic acid (IAA) in 0.1% ethanol treatment of 4 d old Col-0 seedlings


title: AcB61 #12242847


title: AcB55 #12256568b


title: AcB55 #12256487


title: AcB61 #12243313


title: AcB55 #12246570


title: Old Control Day 6 #73 C


title: Old Control Day 6 #71 C


title: Old Treatment Day 30 #97 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 10 #87 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 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 10 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 30 #96 C


title: Old Treatment Day 10 #89 C


title: Old Treatment Day 10 #90 C


title: Old Treatment Day 6 #76 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 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 6 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 30 #99 C


title: Old Treatment Day 30 #98 C


title: Old Treatment Day 6 #80 C


title: Old Treatment Day 10 #88 C


title: Young Treatment Day 30 #49 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 3 #69 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 3 #66 C


title: Old Treatment Day 10 #86 C


title: Young Treatment Day 30 #50 C


title: Young Treatment Day 30 #46 C


title: Young Treatment Day 30 #48 C


title: Old Treatment Day 6 #79 C


title: Old Treatment Day 3 #70 C


title: Young Treatment Day 10 #40 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Old Treatment Day 3 #67 C


title: Old Treatment Day 3 #68 C


title: Old Treatment Day 30 #100 C


title: Young Treatment Day 10 #39 C


title: Old Treatment Day 6 #77 C


title: Young Treatment Day 30 #47 C


title: Young Treatment Day 6 #30 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Young Treatment Day 10 #38 C


title: Young Treatment Day 10 #37 C


title: Young Treatment Day 6 #27 C


title: Young Treatment Day 6 #28 C


title: Young Treatment Day 6 #26 C


title: Young Treatment Day 10 #36 C


title: Young Treatment Day 3 #20 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 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 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Young Treatment Day 0 #9 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 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Young Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Young Treatment Day 3 #16 C


title: Young Treatment Day 0 #6 C


title: Young Treatment Day 3 #17 C


title: Young Treatment Day 0 #7 C


title: Old Treatment Day 0 #59 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 2:
·	Experimental Factors: Age and Atrophy
·	How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
·	Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
·	Experimental Factors: Age, Atrophy, Recovery (Time)
·	How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
·	Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
·	Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2)	Samples used, Extract preparation, and Labeling
Ø	Biosource properties
·	Organism: Rattus Norvegicus
·	Animal Source: Harlan Labs, NIA colony
·	Sex: male
·	Age: 3-4 months or 30-31 months after birth
·	Organism part: soleus skeletal muscle
·	Cell type: mixed in muscle tissue
·	Strain or line: Fischer x Brown Norway F1 rats
·	Genetic variation: N/A
·	Individual genetic characteristics: N/A
·	Disease state or normal: normal
·	State of muscle: Old Day 0 Treatment muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

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


title: Young Treatment Day 3 #19 C


title: Young Treatment Day 0 #8 C


title: Old Treatment Day 0 #58 C


title: Old Treatment Day 0 #60 C


title: Young Treatment Day 3 #18 C


title: Old Treatment Day 0 #57 C


title: Old Treatment Day 0 #56 C


title: Old Control Day 6 #75 A


title: Old Control Day 6 #74 A


title: Old Control Day 6 #75 B


title: Old Control Day 6 #73 A


title: Young Treatment Day 0 #10 C


title: ARPE-19 cells grown on collagen I

description:
I.	Exp Design
1.	Type of experiment: Comparison of native versus cultured RPE cells
2.	Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices
3.	How many hybridizations in exp: 21
4.	If a common reference used for all the hybs: no
5.	Quality control steps: three independent arrays for each condition
6.	Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors.

II.	Samples used, extract prep, and labeling
1.	Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells.
2.	Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin).
3.	Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions.
4.	Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP.
5.	No external controls were added.

III.	Hybridization procedures and parameters
1.	Sample, array type, batch and serial # used
2.	Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with  Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS.
IV.	Measurement data and specifications of data processing
1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis.
3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Keywords = retinal pigment epithelium
Keywords = ARPE-19
Keywords = macula
Keywords = extracellular matrix


title: Old Control Day 6 #72 A


title: Old Control Day 6 #74 B


title: Old Control Day 6 #73 B


title: Old Control Day 6 #71 A


title: Old Control Day 6 #71 B


title: Old Treatment Day 30 #99 A


title: Old Control Day 6 #72 B


title: Old Treatment Day 10 #89 A


title: Old Treatment Day 10 #90 A


title: Old Treatment Day 30 #98 A


title: Old Treatment Day 30 #99 B


title: Old Treatment Day 30 #100 B


title: Old Treatment Day 10 #87 A


title: Old Treatment Day 10 #89 B


title: Old Treatment Day 30 #100 A


title: Old Treatment Day 30 #97 A


title: Old Treatment Day 30 #97 B


title: Old Treatment Day 30 #96 A


title: Old Treatment Day 10 #90 B


title: Old Treatment Day 10 #88 A


title: Old Treatment Day 6 #79 A


title: Old Treatment Day 30 #96 B


title: Old Treatment Day 6 #78 A


title: Old Treatment Day 6 #80 A


title: Old Treatment Day 10 #87 B


title: Old Treatment Day 10 #86 B


title: Old Treatment Day 10 #88 B


title: Old Treatment Day 6 #77 A


title: Old Treatment Day 6 #80 B


title: Old Treatment Day 6 #78 B


title: Old Treatment Day 6 #76 A


title: Old Treatment Day 6 #79 B


title: Old Treatment Day 3 #68 A


title: Old Treatment Day 3 #70 A


title: Old Treatment Day 6 #77 B


title: Old Treatment Day 6 #76 B


title: Old Treatment Day 3 #67 A


title: Young Treatment Day 30 #49 A


title: Old Treatment Day 3 #68 B


title: Old Treatment Day 3 #69 A


title: Young Treatment Day 30 #50 A


title: Young Treatment Day 30 #50 B


title: Old Treatment Day 3 #70 B


title: Young Treatment Day 30 #46 A


title: Young Treatment Day 30 #47 A


title: Young Treatment Day 30 #48 A


title: Old Treatment Day 3 #67 B


title: Young Treatment Day 30 #49 B


title: Young Treatment Day 10 #40 A


title: Old Treatment Day 3 #69 B


title: Young Treatment Day 30 #47 B


title: Young Treatment Day 10 #39 A


title: Young Treatment Day 30 #48 B


title: Old Treatment Day 3 #66 A


title: Old Treatment Day 3 #66 B


title: Young Treatment Day 30 #46 B


title: Young Treatment Day 10 #38 A


title: Young Treatment Day 10 #36 A


title: Young Treatment Day 10 #37 A


title: Young Treatment Day 10 #40 B


title: Young Treatment Day 10 #37 B


title: Young Treatment Day 6 #26 A


title: Young Treatment Day 6 #30 A


title: Young Treatment Day 6 #29 B


title: Young Treatment Day 6 #27 A


title: Young Treatment Day 6 #29 A


title: Young Treatment Day 10 #38 B


title: Young Treatment Day 3 #20 A


title: Young Treatment Day 6 #28 A


title: Young Treatment Day 6 #30 B


title: Young Treatment Day 10 #36 B


title: Young Treatment Day 10 #39 B


title: Young Treatment Day 3 #18 A


title: Young Treatment Day 3 #20 B


title: Old Control Day 6 #72 C


title: Young Treatment Day 3 #16 A


title: Young Treatment Day 3 #19 B


title: Young Treatment Day 3 #18 B


title: Young Treatment Day 6 #27 B


title: Young Treatment Day 6 #28 B


title: Young Treatment Day 3 #19 A


title: Young Treatment Day 6 #26 B


title: Young Treatment Day 3 #17A


title: Young Treatment Day 6 #29 C


title: Young Treatment Day 3 #16 B


title: Young Treatment Day 3 #17 B