Quantitative RT-PCR analysis was performed for selected genes with appropriate primers (Additional file 5: Table S1).
The docking studies were performed for selected compounds (6, 7, 8, 15 and 16) in the binding site of MMP-2 and MMP-9 proteins (PDB entries: 1HOV and 4H3X respectively) using AutoDock Vina [23, 32] and graphical user interface, Auto-dock tools installed on windows 7 [17].
RT-qPCR was performed for selected targets using SYBR Green Master-Mix (Bio-Rad) to validate the microarray results.
Quantitative RT-PCR was performed for selected genes in order to validate the results obtained in the microarray study.
The Solvent Accessibility, which calculates the solvent accessible surface of protein residues, was performed for selected amino acid residues without water.
The validation of gene chip analysis was performed for selected genes by FACS staining (Figure 3A) and real-time RT PCR (Figure 3B).
Immunostainnings for EMA (1∶1, clone E29, DAKO), GFAP (1∶200, clone 6F2, DAKO), OLIG2 (1∶100, RnD systems, Abingdon, UK) and Neurofilament Protein 70 (1∶50, clone 2F11, DAKO) were performed for selected cases.
In situ hybridization or immunocytochemistry was performed for selected genes from each "Adeno" cluster and confirmed that the enhanced gene expression was indeed derived from the neoplastic cells (Figure 3).
Conventional ELISA (Table S2) was performed for selected cytokines on pooled samples from an additional 29 keratoconus patients (76% male, mean age = 38±10) and 38 control individuals (52% male, mean age = 40±12).
For validation of microarray results, comparative qPCR was performed for selected genes on randomly chosen NZM cell lines, with quantification cycle (Cq) values normalised to total RNA input and converted to quantities relative to the cell line with the lowest abundance of a given gene by using the delta-Cq method.
