4.6. The Human ECM and Adhesion Molecules RT2 Profiler PCR Gene Array

The expression of genes was elucidated using the RT2 Profiler PCR Array for 84 human ECM and cell adhesion genes (Qiagen, Chadstone, VIC, Australia). This array detects gene expressions that encode human ECM proteins, including transmembrane receptors, adhesion molecules, E-cadherin, ECM adhesion, basement membrane constituents, collagen, ECM structural constituents, ECM proteases and protease inhibitors. The array contains a panel of 5 housekeeping genes used for normalisation of data, plus a panel of three reverse transcription controls, a genomic DNA control and three positive PCR controls.

In this experiment, human fibroblasts cells were seeded to a density of 3 × 10⁶ cells in a 40 mL flask containing 12 mL of complete media and incubated for 48 h to produce a confluent monolayer. The fibroblasts were supplemented as follows: (1) 5% LR-PRP; (2) 5% LP-PPP; and (3) serum-free media (negative control). Following 12 h incubation, fibroblast cells were extracted by standard laboratory enzymatic methods [98]. Each cell pellet was individually collected for fibroblast mRNA extraction using the RNAeasy mini kit (Qiagen) exactly according to the manufacturer’s instructions. Thereafter, RNA samples were used to assess the RNA quality and concentration using Nanodrop (Denovix, Melbourne, Australia). The remaining RNA were aliquoted (6 µL RNA) into individual 0.5 mL micro-tubes for mRNA expression analysis according to the method previously described by Dargahi [99].