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Sample GSM210290 Query DataSets for GSM210290
Status Public on Jul 16, 2007
Title Subject code A44
Sample type RNA
 
Source name JA
Organism Homo sapiens
Characteristics Age: 20
Gender: M
Gender: M
Chip ID: 1375488010
Position: C
Sample Group: Y
Treatment protocol n/a
Growth protocol n/a
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from human muscle with TRIzol reagent (Invitrogen, Carlsbad, CA). Briefly, 25–50 mg of muscle was homogenized in 1 ml of TRIzol reagent at 4°C, left at room temperature for 5–10 min, followed by addition of 0.2 ml of chloroform, vortexing for 15 s, and centrifugation @ 12,000 rpm @ 4°C for 15 min. The supernatant was transferred to a fresh tube and mixed with 0.5 ml of isoproponal ethanol, stood at ~22°C for 10 min, and centrifuged at 12,000 rpm at 4°C for 10 min. The RNA pellet was washed twice with 0.5 ml of 75% ethanol, air dried and dissolved in 14 µl of Depc-treated ddH2O with 2 µl aliquots stored at −80°C. The concentration and purity of the RNA was determined using a UV spectrophotometer (Shimadzu UV-1201; Mandel Scientific, Guelph, Ontario) at the absorbance of 260/280 nm. Measurements were done in duplicate and had an average coefficient of variation (CV) of <10%. The average purity (OD260/OD280) of the samples was >1.5 before DNAase treatment. RNA integrity was assessed in a randomly chosen subset of samples using agarose gel electrophoresis, and the OD ratio of 28S to 18S rRNA was consistently greater than 1 for each sample.
Label biotin
Label protocol The resulting total RNA samples were further assessed for integrity prior to chipping using a Nanodrop Spectrophotometer and the Agilent Bioanalyzer Nano Chip System. Samples which passed this initial quality control assurance step were then amplified one round, using an RNA Amplification Kit (Ambion) according to the manufacturer's instructions. The resulting purified cDNA product of was resuspended in 18 µl and dried down using a speedvac. This was then converted to cRNA using an in vitro transcription kit according to the manufacturer's instructions (Roche), and again assessed for quality by using the Nanodrop and Bioanalyzer
 
Hybridization protocol Labeled cRNA samples that passed this second round of quality control were then hybridized to Human Ref-8 BeadChips (Illumina) according to the manufacturer's instructions (approximately 23,000 genes), using equipment specified by the manufacturer (Illumina). Briefly, 850 ng biotin-labeled cRNA in 11.3 µl nuclease-free water was adjusted to 34 µl through the addition of 22.7 µl of 5:3 HybE1 buffer/formamide. The sample was heated at 65°C for 5 min, allowed to cool to room temperature, and then immediately added to a single array of an 8-array Human Ref-8 BeadChip. Once all 8 samples were added to each BeadChip, it was sealed in a Hyb Cartridge and incubated for 16 h at 55°C with rotation in an Illumina hybridization oven (rotation setting 5). Following overnight hybridization, BeadChips were moved to a slide rack and serially washed using gentle rotation in glass staining dishes filled with a) 250 ml Illumina Wash Buffer×5 min, b) 250 ml 100% ethanol×10 min, c) 250 ml Illumina Wash Buffer×2 min. BeadChips were then blocked for 10 min in 4 ml Block E1 buffer (Illumina), followed by staining for 10 min in 1 µg/ml Streptavidin-Cy3 conjugate (GE Healthcare) in Block E1 buffer. Stained BeadChips were finally washed using gentle rotation in a glass staining dish filled with 250 ml Illumina Wash Buffer×5 min. BeadChips were dried by centrifugation at 280×g for 4 min and stored in a light-tight box until reading.
Scan protocol Processed arrays were read using a BeadStation array reader (Illumina) according to the manufacturer's instructions. Scan settings were for single color (green) scanning: Factor = 2.506, PMT = 561, Filter = 100%.
Description none
Data processing Raw data (see related publication for normalization procedure)
 
Submission date Jul 13, 2007
Last update date Jul 16, 2007
Contact name Serban Ciotlos
E-mail(s) smelov@buckinstitute.org
Phone 4085068553
Organization name Buck Institute for Research on Aging
Lab Melov Lab
Street address 8001 Redwood Blvd
City Novato
State/province CA
ZIP/Postal code 94945
Country USA
 
Platform ID GPL2700
Series (1)
GSE8479 Resistance Exercise Reverses Aging in Human Skeletal Muscle

Data table header descriptions
ID_REF
VALUE raw signal
Detection Pval

Data table
ID_REF VALUE Detection Pval
GI_10047089-S 8859.116 0
GI_10047091-S 87.72762 0.2541935
GI_10047093-S 568.4612 0
GI_10047099-S 168.4102 0.003870968
GI_10047103-S 850.8594 0
GI_10047105-S 90.71096 0.1845161
GI_10047121-S 76.49963 0.6774194
GI_10047123-S 119.9471 0.01806452
GI_10047133-A 82.34085 0.4387097
GI_10047133-I 86.16744 0.2929032
GI_10092578-S 83.17556 0.3987097
GI_10092585-S 135.6537 0.01032258
GI_10092596-S 199.6423 0.003870968
GI_10092600-S 446.4234 0
GI_10092602-S 74.50375 0.7677419
GI_10092603-S 92.13731 0.1522581
GI_10092611-A 135.581 0.01032258
GI_10092616-S 78.70596 0.5909677
GI_10092618-S 2140.211 0
GI_10092638-S 182.0234 0.003870968

Total number of rows: 24354

Table truncated, full table size 734 Kbytes.




Supplementary data files not provided
Processed data not provided for this record

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