|
| Status |
Public on Dec 31, 2015 |
| Title |
BA11 sample #177 |
| Sample type |
RNA |
| |
|
| Source name |
Brain prefrontal cortex, broadman areas 11
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
age: 53
pmi: 21.3
ph: 6.2
rin: 7.3
Sex: M
race: W
tod: 6.280881442
|
| Treatment protocol |
not applicable
|
| Growth protocol |
not applicable
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen samples stored in TRIzol (Invitrogen, Carlsbad, Calif.) and processed for microarray analysis according to the microarray manufacturer’s protocol (Affymetrix, Santa Clara, Calif.)
|
| Label |
biotin
|
| Label protocol |
According to standard affymetrix protocol: • 10ul Purified cDNA10ul • DEPC H2O • 4ul 10x IVT Labeling Buffer • 12ul IVT Labeling NTP mix • 4ul IVT Labeling Enzyme mix - Incubate at 37C 16 hour in an incubating oven or thermal cycler with heated lid to avoid condensation. - Purify labeled cRNA generated from either IVT labeling reaction using RNeasy RNA Purification Mini kit (Qiagen) using the RNA Cleanup Protocol according to manufacturers instructions. - Quantitate Biotin labeled cRNA using 260nm/280nm spectrophotometric assay.
|
| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix hybridization kit materials: • Heat the hybridization cocktail to 99°C for 5 minutes in a heat block • Incubate the probe array filled with Pre-Hybridization Mix at 45°C for 10 minutes with rotation • Transfer the hybridization cocktail that has been heated at 99°C, to a 45°C heat block for 5 minutes • Spin the hybridization cocktail at maximum speed in a microcentrifuge for 5 minutes to collect any insoluble material from the hybridization mixture • Remove the array from the hybridization oven. Vent the array with a clean pipette tip and extract the Pre-Hybridization Mix from the array with a micropipettor. Refill the array with the appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube • Place probe array into the hybridization oven, set to 45°C • To avoid stress to the motor, load probe arrays in a balanced configuration around the axis. Rotate at 60 rpm • Hybridize for 16 hours • During the latter part of the 16-hour hybridization, proceed to the GeneChip® Expression Wash, Stain and Scan User Manual, P/N 702731, to prepare reagents for the washing and staining steps required immediately after completion of hybridization
|
| Scan protocol |
Affymetrix GeneChIP Scanner 3000 7G
|
| Description |
dissected from frozen coronal blocks ~2–3 cm caudal to the temporal pole
|
| Data processing |
All samples were analyzed using the Affymetrix Human Gene 1.1 ST microarray platform. Gene expression values were corrected, quantile-normalized, and log2-transformed via Affymetrix Expression Console software (build 1.2.1.20) using RMA algorithm. A total of 33,297 probe sets were available on each array; only those probe sets with annotated gene symbols were selected (20,237 gene symbols). If a gene was represented by multiple probe sets, the one with the largest intensity interquartile region was selected to represent that gene. Microarray data for each brain region were analyzed separately.
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| |
|
| Submission date |
Jul 31, 2015 |
| Last update date |
Dec 31, 2015 |
| Contact name |
Etienne Sibille |
| E-mail(s) |
[email protected]
|
| Phone |
412-624-0804
|
| Organization name |
University of Pittsburgh
|
| Department |
Psychiatry
|
| Street address |
450 Technology Drive, Suite 231
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15219 |
| Country |
USA |
| |
|
| Platform ID |
GPL11532 |
| Series (1) |
| GSE71620 |
The effects of aging on circadian patterns of gene expression in the human prefrontal cortex |
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