ID_REF = VALUE = Agilent Feature Extraction Software (v 184.108.40.206) was used for background subtraction and LOWESS normalization.
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA).
All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3 (Agilent Technology, USA). The averages of normalized ratios were calculated by dividing the average of normalized signal channel intensity by the average of normalized control channel intensity. Functional annotation of genes was performed according to Gene OntologyTM Consortium (http://www.geneontology.org/index.shtml) by GeneSpringGX 7.3. Gene classification was based on searches done by BioCarta (http://www.biocarta.com/), GenMAPP (http://www.genmapp.org/), DAVID (http://david.abcc.ncifcrf.gov/), and Medline databases (http://www.ncbi.nlm.nih.gov/).
After checking labeling efficiency, fragmentation of cRNA was performed by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60oC for 30 min. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto assembled Agilent’s Canine Oligo Microarray (44K). The arrays hybridized at 65oC for 17 hours using Agilent Hybridization oven (Agilent Technology, USA). The hybridized microarrays were washed as the manufacturer’s washing protocol (Agilent Technology, USA).
Amplified and labeled cRNA was purified on cRNA Cleanup Module (Agilent Technology) according to the manufacturer’s protocol. Labeled cRNA target was quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
Immediately after the teeth were extracted, dental pulp tissues were collected from vertically cut tooth 1mm under the cemento-enamel junction. Then tissues from each tooth were cut into small pieces for cell culture in medium made by mixing Dulbecco's modified Eagle's medium (Gibco BRL, Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (Gibco BRL, Life Technologies, Grand Island, NY, USA) and antibiotics, consisting of penicillin-G (100U/ml), streptomycin (100 ㎍/ml), Fungizone (0.25 g/ml) (Gemini Bio-Products, Inc., Woodland, CA, USA), in a humidified atmosphere of 5% CO2 at 37℃. Dental pulp cells out-grown from the tissues were subcultured and grown to confluence. Then subcultures were successively passed at 1:2 ratio until three passages. Dental pulp cells that had been cultured through three passages were used in the experiments.
For rapid freezing, the premolars were placed in cryopreservation vials containing 10% dimethyl sulfoxide (Me2SO: Sigma Chemical, St. Louis. MO, USA) within 5 minutes and stored in a liquid nitrogen at -196℃ for 1 week. Then thawing and culture method was same with the slow freezing group. For slow freezing treatment, the premolars were placed in cryopreservation vials containing 10% dimethyl sulfoxide (Me2SO: Sigma Chemical, St. Louis, MO, USA) within 5 minutes. For a constant cooling rate, they were placed in cell freezer container with 100% isopropanol and stored in a -80℃ deep freezer for twenty-four hours and they stored in liquid nitrogen at -196℃ for 1 week then thawed in a water bath rapidly at 37℃ for 3 minutes. For the experiment, dental pulp cells out-grown from the tissues had been cultured in the same way with control group.
Total RNA extracted using Trizol following manufacturer's instructions.