PCR array Sample Clauses

PCR array miScript xxXXX PCR Array kit, format E, 384-xxxxx, from Qiagen was used according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Osteogenic, adipogenic and osteo/adipogenic clonally derived MSC cDNA were used as sample of interest and non- differentiating clone as control in the array. The cDNAs for this experiment were generated by using miScript II RT Kit (Qiagen, Hilden, Germany, cat no:218161) with miScript HiSpec Buffer from frozen RNAs which was kindly given to us by Xx. Xxxxxxx. These RNAs were extracted from single clones MSCs which were established by limiting dilution of cell suspensions of MSC cultures. The multipotent differentiation capacity to osteogenic or adipogenic linages, bipotential (osteogenic and adipogenic) and non- differentiated colons were determined by morphological examination and gene expression analysis of the osteogenic (Runx2 and ALP) and adipogenic (PPARγ and LPL) markers by qRT-PCR. To prepare master mix, according to the manufacturer’s instructions, 2050μl QuantiTect SYBR Green PCR Master Mix and 410 μl miScript Universal Primer, 1540 µl RNase-free water and 0.5–1 ng cDNA per well were gently mixed and then 10 μl of master mix applied per well. The plate was centrifuged for 1 min at 1000 g at room temperature. The details of thermal profile using the Bio-Rad system is shown below (Table 2-3) Cycles Step Duration of cycle Temperature 1 Initial activation step 15 minutes 95°C 40 Denaturation 15 seconds 94°C Annealing 30 seconds 55°C Extension 30 seconds 70°C Table 2-3; Thermal profile cycles used for PCR array The data were analysed automatically using the ∆∆Ct method, using data analysis software which is available at xxxx://xxxxxxxxxxxxxxx.xxxxxxxxxxxxx.xxx/xxxxx. In this format, C. elegans xxXXX-39 miScript Primer Assay, 3 sno/snRNA miScript PCR Control (SNORD61/95/96A), miRTC miScript Primer Assay AND positive PCR control (PPC) were used as reference RNAs.