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: [email protected] · 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 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
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
