Real-time PCR focused-gene array profiling of gingival and periodontal ligament fibroblasts.
The techniques for the establishment of primary gingival and periodontal ligament fibroblast cultures have been well established for over 30 years. It is only more recently, with the commercial availability of real-time PCR (RT-PCR) gene arrays that the expression profiles of up to 84 genes can be carried out simultaneously. Each focused panel of genes can identify the up- or down-regulation of genes associated with any one of over 100 biological pathways or specific disease states. Fibroblasts for RNA extraction and subsequent gene expression analysis can be collected under various experimental conditions and stored in RNA-preserving solution (e.g., RNAlater) for processing at a later date or extracted immediately. The "gold standard" method for the extraction of RNA from fibroblasts for RT-PCR purposes is the TRIzol reagent method. With the addition of a spin-column clean-up step, any phenol carried over from the TRIzol step is removed, thus ensuring a high yield of quality RNA. The RNA is then reverse transcribed to cDNA and analyzed using the RT-PCR focused-gene arrays. Data analysis is made easy using on-line array analysis software packages.
Chou
Patty
P
Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand.
Milne
Trudy J
TJ
D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D010513:Periodontal Ligament / Q000166:cytology*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*