strain: Sprague-Dawley tissue: proximal tibial epiphysis number of tissues: 4 from animals 1 and 2 age: 10 days postnatal zone: resting zone
Treatment protocol
Proximal tibial epiphyses of 10-day-old Sprague-Dawley rats (n=8) were rapidly excised, trimmed of any soft connective tissues, embedded in Tissue-Tek O.C.T. Compound, sectioned (60 μm thick), mounted on Superfrost Plus slides, and stored at -80°C for subsequent processing. Slides were thawed for 15 s and then placed in 70% ethanol, fixed in 100% methanol, washed in 95% ethanol, stained in eosin (0.2% eosin, 0.5% acetic acid, 75% ethanol), washed in 70% ethanol, dehydrated in 100% ethanol, and placed in xylene (each step for 1 min, at room temperature). Microdissection was performed using an inverted microscope, razor blades, and hypodermic needles. Tissues were microdissected in a xylene droplet and collected in 10 μl of 4M guanidine thiocyanate, 25mM sodium citrate pH7, and 0.1M β-mercaptoethanol on ice. Enzymatic digestion of microdissected tissues was performed using buffered proteinase K solution (1,500 μl DEPC-treated water, 30 μl 1M Tris-HCl pH7, and 57.5 μl proteinase K (20 mg/ml in glycerol)).
Growth protocol
Animals were maintained under standardized conditions and handled in accordance with the Animal Ethics Committee of Northern Stockholm.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using phenol:chloroform:isozmyl alcohol (25:24:1), precipitated in 3M NaOAc and then 8M LiCl, and washed with 75% ethanol. For the complete protocol please refer to Nilsson et al. J Endocrinol April 1, 2007 193 75-84 (doi: 10.1677/joe.1.07099).
Label
biotin
Label protocol
ss cDNA was prepared with the Ambion WT Expression kit from 100ng of total RNA
Hybridization protocol
Following fragmentation and biotinylation (Affymetrix Gene Chip WT Labeling Kit), 5 ug of ss cDNA were hybridized for 16 hr at 45C on GeneChipRat 1.0 STArray. They were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
Description
RZ #1
Data processing
The data were analyzed with Partek Genomics Suite 6.6 using robust multi-array average (RMA) analysis, which adjusts for background noise on each array using only the PM probe intensitites and subsequently normalizes data across all arrays using quantile normalization followed by median polish summarization to generate a single measure of expression. These expression measures were then log transformed (base 2) and lists of spatially regulated genes were generated.
