Rt2 pcr profiler array real time pcr

Total RNA was isolated from periventricular tissue using the acid guanidinium phenol chloroform method and RNeasy Mini Kit supplied by QIAGEN (Germantown, MD, USA). The OD ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to manufacturer on 0.1-1 μg total RNA using iScript^TM cDNA Synthesis Kit (Bio-Rad, CA, USA) and RT² First Strand Kit (QIAGEN). RT² PCR Profiler Array real-time PCR (QIAGEN custom made, Cat no. CAPN11841 and Wound healing, Cat no. 330231 PANZ-121ZA) were used to quantify the mRNA expression of toll-like receptor (TLR)-4, monocyte chemoatractant protein (MCP)-1, interleukin (IL)-1β, IL-6, IL-8, IL1 receptor (R)1, tumor necrosis factor (TNF) α, matrix metalloprotease (MMP) 9, and heme oxygenase (HO)-1. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, custom made by QIAGEN, Cat no. CAPN11841). The fold change values were calculated by normalizing against Sham Control samples from untreated animals. Data are presented as box plots, displaying medians and 25^th and 75^th percentiles. Expression was analyzed using RT² SYBR Green Fluor qPCR Mastermix (QIAGEN). Amplification was performed as described by the manufacturer (QIAGEN) for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data analyzed using iCycler iQ Optical System Software (Bio-Rad).

Total RNA was isolated from choroid plexus and HCPEpiC cells using the acid guanidinium phenol chloroform method and RNeasy Mini Kit supplied by QIAGEN (Germantown, MD, USA). The optical density (OD) ratio (OD at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to manufacturer on 0.1 to 1 μg total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and RT² First Strand Kit (QIAGEN, Germantown, MD, USA). RT² PCR Profiler Array real-time PCR (custom made by QIAGEN, Germantown, MD, USA) were used to quantify the mRNA expression of TLR-4, IL1R1, FAS, NF-Κβ, MCP-1, IL-8, IL-1β, TNFα, IL-6 and HO-1. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, custom made by QIAGEN; Germantown, MD, USA). The fold change values were calculated by normalizing against control samples from untreated animals or cells. Data are presented as box plots, displaying medians and 25^th and 75^th percentiles, for in vivo data and as bars, displaying mean ± SEM, for in vitro data. Expression was analyzed using RT² SYBR Green Fluor qPCR Mastermix (QIAGEN, Germantown, MD, USA). Amplification was performed as described by the manufacturer (QIAGEN) for 40 cycles in an iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA) and data analyzed using iCycler iQ Optical System Software (Bio-Rad, Hercules, CA, USA).