|Application:||Gene expression microarray analysis|
|Number of samples:||8|
|Release date:||Apr 17 2009|
|Last update date:||Jul 31 2017|
|Taxon:||Homo sapiens, Rattus norvegicus|
|Dataset link||Gene Expression Changes in Response to UV Inactivated Baculoviral Vector Transduction of the Rat Brain In Vivo|
For in vivo viral inoculation, adult male Wistar rats (weighing 250–300 g) were used. Five μl of baculovirus vectors (5 x 107 viral particles) was injected stereotaxically into the striatum at two injection sites (AP ±1.0mm, ML +2.5mm, and DV -5.0mm from bregma and dura) using a 10 μl Hamilton syringe connected with a 30-gauge needle, at a speed of 0.5 μl/min. The needle remained in place for another 5 min before being slowly retracted. Rats were sacrificed and the brain tissues were collected 48 hours later for analysis. Five μl of PBS was injection into the contralateral striatum and the tissues were collected and used as controls. Seven rats were used for the study. Total RNA was isolated using Trizol® (Invitrogen, Carlsbad,CA) according to the manufacturer’s instruction. RNA was further cleaned up using RNeasy mini kit (Qiagen). Total RNA was quantified using a Nanodrop spectrophotometer and its quality was further assessed using an RNA nanochip on a Bioanalyzer. One to 10 ug of good quality RNA (i.e. A260/A280 > 2.0 and A230/A260 > 2.1) was used to prepare labeled cRNA using either Affymetrix’s One-Cycle Target Labeling and Control Reagents or Kreatech’s RNA ampULSe amplification and labeling kit (Kreatech, Amsterdam, The Netherlands). Hybridization mixes with 15 ug of fragmented cRNA previously checked on the bioanalyzer were prepared according to Affymetrix’s GeneChip® Hybridization, Wash, and Stain Kit’s instruction and used to hybridize the human GeneChip Human Genome U133 Plus 2.0 or GeneChip Rat Genome 230 2.0 for 16 hours at 45ºC. The arrays were washed and stained with phycoerythrin according to the manufacturer’s instructions. Chips were scanned using GeneChip® Scanner 3000 and data acquired using the GCOS software. Data obtained from GCOS was exported using the Data Transfer Tool and transferred to GeneSpring and Genedata software respectively. The GC-RMA preprocessor file was used to re-process imported CEL files and to normalize the data at the probe level. Replicates (at least 3 per experiment) were grouped and data normalized using the default setting of Genespring (Data transformation: values less than 0.01 to 0.001; Global normalization function: Per Chip normalization to median or a percentile; Gene normalization function: normalize to median was used. The Genedata’s Refiner function was used to assess the overall quality of the hybridization. To analyze the data, the genes were grouped according to the cells’ or brain’s treatment (Non- infected/infected) and the log of normalized values were taken for further analysis. Data was filtered according to their flags in such a way that at least 5 out of 7 brain samples or 2 out of 3 cell replicates had a present call and that at least one of the groups of replicates (non-infected/infected) fulfilled this condition. The volcano plot was sometime used to filter the data further with the following cut-off (2-fold difference in expression level and t-test p-value of less than 0.05). Further data manipulation for comparison purposes are described in the text.