Odontoblasts obtained from third molars of young patients and tissue cultured for 1h as controls without TGF-beta.
Treatment protocol
Fresly extracted human third molars were cut 2-3mm apically from cement-enamel junction and pulp tissues were removed. The crowns were embedded into agarose gel pulp chambers facing upwards, and the pulp chambers were filled with OPTIMEM. Odontoblasts were cultured for 1h without TGF-beta.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol-protocol and further purified by Qiagen RNeasy Mini Kit
Label
biotin
Label protocol
Double-stranded DNA was synthesized by means of the Superscript Choice System (Gibco BRL Life Technologies, Rockville, MD, USA) and T7-(dT)24 primer. The DNA was purified using GeneChip Sample Cleanup Module (Qiagen, Valencia, CA, USA). In vitro transcription was performed to produce biotin labeled cRNA using a BioArray HighYield RNA Transcription Labeling Kit (Enzo Diagnostics, Farmingdale, NY, USA) according to the manufacturer’s instructions. Biotinylated cRNA was cleaned with an GeneChip Sample Cleanup Module (Qiagen), fragmented to 35 to 200 nt, and hybridized to Affymetrix HGU133plus 2.0 (odontoblast samples). After being washed, the array was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR, USA). Staining signal was amplified by biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA, USA) and second staining with streptavidin-phycoerythrin.
Hybridization protocol
Experimental procedures for GeneChip (Affymetrix, Santa Clara, CA, USA) were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual.
Scan protocol
HP GeneArray Scanner was used.
Description
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Data processing
The expression data was analyzed using Affymetrix GeneChip Operating Software version 1.1.1. Signal intensities of all probe sets were scaled to the target value of 500.
