Femke Mathot

Chapter 9 148 expression of platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) and vascular endothelial cell growth factor alpha (VEGF1). Establishment of the ECM was assessed by monitoring expression of collagen type I (COL1A1), collagen type III (COL3A1), Fibulin 1 (FBLN1) and laminin subunit beta 2 (LAMB2). Immunoglobulin expression was determined by quantification of cluster of differentiation 96 (CD96), a cell surface marker. Both Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and AKT Serine/Threonine Kinase 1 (AKT1) were used as reference genes. Expression of the AKT1 gene was determined to be more stable and considered the most optimal housekeeping gene based on BestKeeper analysis and therefore was used as reference gene in the analysis. 28 The 2-ΔΔCT method was used to calculate the mRNA levels which were expressed as a ratio of AKT1. 29-31 Primer sequences for each of the genes that were analyzed are displayed in table 1 . Table 1. mRNA primer sequences. ECM = extracellular matrix. NGF = nerve growth factor, GDNF = glial cell line-derived neurotrophic factor, PTN = pleiotrophin, GAP43 = growth associated protein 43, BDNF = brain-derived neurotrophic factor, PMP22 = peripheral myelin protein 22, MPZ = myelin protein zero, VEGF-a = vascular endothelial cell growth factor alpha, PECAM = platelet endothelial cell adhesion molecule 1, COL1A1 = collagen tpe 1, COL3A1 = collagen type III, FBLN1 = fibulin 1, LAMB2 = laminin beta 2 subunit, CD96 = cluster of differentiation 96, AKT1 = threonine kinase 1, GAPDH = glyceraldehyde-3-phosphate dehydrogenase. Gene ID Biology Forward primer Reverse primer NGF Neurotropic marker ATACAGGCGGAACCACACTCAG ATACAGGCGGAACCACACTCAG GDNF Neurotropic marker CACCAGATAAACAAATGGCAGTGC CACCAGATAAACAAATGGCAGTGC PTN Neurotropic marker ACTGGAAGTCTGAAGCGAGC CTTCTTCTTAGATTCTGCTTGAGGT GAP43 Neurotropic marker GTCCACTTTCCTCTCTATTTC TGTTCATTCCATCACATTGA BDNF Neurotropic marker AGAGGCTTGACATCATTGGCTG CAAAGGCACTTGACTACTGAGCATC PMP22 Myelination marker GTTAAAGGGAACGCCAGGA AGTTTCTGCAGCCCAAAGGA MPZ Myelination marker GAGGAGGCTCAGTGCTATGG GCCCGCTAACCGCTATTTCT VEGF-a Angiogenic marker ATCTGCATGGTGATGTTGGA GGGCAGAATCATCACGAAG PECAM/ CD31 Angiogenic marker AACAGTGTTGACATGAAGAGCC AACAGTGTTGACATGAAGAGCC COL1A1 ECM protein GTAACAGCGGTGAACCTGG CCTCGCTTTCCTTCCTCTCC COL3A1 ECM protein TTGAAGGAGGATGTTCCCATCT ACAGACACATATTTGGCATGGTT FBLN1 ECM protein AGAGCTGCGAGTACAGCCT CGACATCCAAATCTCCGGTCT LAMB2 ECM protein ACACGCAAGCGAGTGTATGA AATCACAGGGCAGGCATTCA CD96 Cell surface marker / immunoglobulin AGATTGTGTGATGAAGGACATGG AGATTGTGTGATGAAGGACATGG AKT1 Household gene ATGGCGCTGAGATTGTGTCA CCCGGTACACCACGTTCTTC GAPDH Household gene CCCGGTACACCACGTTCTTC TGTGGTCATGAGTCCTTCCA