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Sample GSM4977 Query DataSets for GSM4977
Status Public on Aug 18, 2003
Title Young Control Day 0 #2 B
Sample type RNA
 
Source name soleus muscle
Organism Rattus norvegicus
Extracted molecule total RNA
 
Description II. Experiment Description
1) Experimental Design
· Authors: J. Scott Pattison, Thomas Ellis Childs, Lillian Folk, Richard Madsen, and Frank W. Booth.
· Laboratory: Dr. Frank Booth, University of Missouri-Columbia, 1600 E. Rollins St, Columbia, Mo, 65211
· Contact: boothf@missouri.edu
· Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 1:
· Experimental Factors: Age
· How many hybridizations in the experiment: 45 unique samples were hybridized once per array x 3 arrays each = 135 separate hybridizations in total.
· Experiment Description: To elucidate the differences in gene expression between young control soleus muscles and old control soleus muscles.
· Type of experiment: control young muscle (3-4 months) vs control old muscle (30-31 months).
Experiment 2:
· Experimental Factors: Age and Atrophy
· How many hybridizations in the experiment: 20 unique samples were hybridized once per array x 3 arrays each = 60 separate hybridizations in total.
· Experiment Description: To elucidate the differences in gene expression between young and old soleus muscles with and without immobilization.
· Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months).
Experiment 3:
· Experimental Factors: Age, Atrophy, Recovery (Time)
· How many hybridizations in the experiment: 95 unique samples were hybridized once per array x 3 arrays each = 285 separate hybridizations in total.
· Experiment Description: Time course of skeletal muscle recovery following atrophy, comparing young and old responses vs. controls.
· Type of experiment: control young muscle (3-4 months) vs immobilized young muscle (3-4 months) vs. control old muscle (30-31 months) vs. immobilized old muscle (30-31 months) at each of recovery times 0, 3, 6, 10, and 30 days.
2) Samples used, Extract preparation, and Labeling
Ø Biosource properties
· Organism: Rattus Norvegicus
· Animal Source: Harlan Labs, NIA colony
· Sex: male
· Age: 3-4 months or 30-31 months after birth
· Organism part: soleus skeletal muscle
· Cell type: mixed in muscle tissue
· Strain or line: Fischer x Brown Norway F1 rats
· Genetic variation: N/A
· Individual genetic characteristics: N/A
· Disease state or normal: normal
· State of muscle: Young Day 0 Control muscle
*The strain of rat used has a propensity for increased longevity over most other strains, due to a failure to develop premature diseases including cancers that shorten the life of other rat strains.

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

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

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

Ø Measurement data and specifications of data processing
· Scanning: The scanning protocols are available at (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
· Raw Image Analysis/Quality Control: After scanning, array images were assessed to confirm scanner alignment. The corners were examined by eye for proper grid alignment. Images were also scanned for the absence of bubbles, scratches, and speckling. If any images with small scratches or speckles that skewed >25% of a given feature, that feature was masked and not quantitated in future analyses. 3'/5' ratios for GAPDH and beta-actin were confirmed to be ~1.0. From the QC report, spike controls were found to be present on all chips, with BioB, BioC, BioD and CreX being found present in increasing intensity.
· Scaling/Normalization: All data were scaled to a target intensity of 1500 (using Affymetrix MAS 5.0 array analysis software), scaling factors for all arrays were within acceptable limits, as were background, and mean intensities. No further normalization was applied.
· Data Analysis:
· Experiment 1:
· Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed. The signal values for all sufficiently detected probe sets were analyzed for significant differences via a Student’s t-test. A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 (raw p=4.478x10-6) were considered significant.
· The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
· Experiment 2:
· Only those probe sets found to be called “present” or “marginal” in > 60% of all samples in an age group were statistically analyzed. The signal values for all sufficiently detected probe sets were analyzed for significant differences via a 2-way ANOVA. A Bonferroni adjustment was applied to all data to control for multiple testing, where a Bonferroni-adjusted p<0.05 were considered significant.
· The final list of statistically changing genes can be found at (Supporting Data to be linked to journal web site upon paper acceptance)
· Experiment 3:
· Only those probe sets found to be called “present” or “marginal
 
Submission date Mar 21, 2003
Last update date Oct 28, 2005
Contact name Scott Pattison
E-mail(s) pattisonj@missouri.edu
Phone 573.882.0820
Organization name University of Missouri
Street address
City Columbia
State/province MO
ZIP/Postal code 65211
Country USA
 
Platform ID GPL86
Series (1)
GSE353 Effect of age on skeletal muscle

Data table header descriptions
ID_REF
Stat_Pairs Affymetrix MAS 5.0 The number of probe pairs in the probe set
Stat_Pairs_Used Affymetrix MAS 5.0 The number of probe pairs in the probe set used in the Detection call
VALUE Affymetrix MAS 5.0 A quantitative measure of the relative abundance of a transcript
ABS_CALL Affymetrix MAS 5.0 A qualitative measurement indicating if the transcript is detected (Present), not detected (Absent), or marginally detected (Marginal)
Detection_p-value Affymetrix MAS 5.0 A p-value indicating the significance of the Detection call

Data table
ID_REF Stat_Pairs Stat_Pairs_Used VALUE ABS_CALL Detection_p-value
AFFX-MurIL2_at 20 20 8 A 0.772364
AFFX-MurIL10_at 20 20 10.6 A 0.737173
AFFX-MurIL4_at 20 20 7.4 A 0.544587
AFFX-MurFAS_at 20 20 18 A 0.205732
AFFX-BioB-5_at 20 20 260.8 P 0.020022
AFFX-BioB-M_at 20 20 389.7 P 0.002556
AFFX-BioB-3_at 20 20 177.4 P 0.021902
AFFX-BioC-5_at 20 20 1131.7 P 0.000095
AFFX-BioC-3_at 20 20 625.6 P 0.000147
AFFX-BioDn-5_at 20 20 727.1 P 0.000857
AFFX-BioDn-3_at 20 20 4464.9 P 0.000195
AFFX-CreX-5_at 20 20 6251.2 P 0.000044
AFFX-CreX-3_at 20 20 9197.9 P 0.000044
AFFX-BioB-5_st 20 20 49.4 A 0.262827
AFFX-BioB-M_st 20 20 42.6 A 0.544587
AFFX-BioB-3_st 20 20 21.8 A 0.804734
AFFX-BioC-5_st 20 20 15.2 A 0.804734
AFFX-BioC-3_st 20 20 13.1 A 0.544587
AFFX-BioDn-5_st 20 20 27.6 A 0.574038
AFFX-BioDn-3_st 20 20 25.3 A 0.354453

Total number of rows: 8792

Table truncated, full table size 318 Kbytes.




Supplementary data files not provided

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