tissue: brain age: 65 pmi: 21.2 ph: 7 rin: 9 Sex: M race: W tod: 6.389433769
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.
