Rt2 sybr green fluor qpcr mastermix

Tissue samples were flash-frozen and immediately used for RNA extraction. Approximately 20 mg of tumor sample was used for RNA extraction. Samples were homogenized using a bullet blender in 600 µL of buffer RLT (RNeasy mini kit) with β-mercaptoethanol. The lysates were centrifuged for 3 min at full speed and the supernatant was used for RNA extraction using RNeasy mini kit (QIAGEN). On column DNA digestion was done using RNase-Free DNase set (QIAGEN). RNA quality and quantity was determined using a spectrophotometer at 260 and 280 nm (Nanodrop-2000, Thermo Scientific). cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies). Quantitative-PCR was done using RT2 SYBR Green Fluor qPCR Mastermix and RT2 qPCR primers (Qiagen) on a CFX96 Touch Real-Time PCR Detection System (Bio Rad). KLK2 (hK2), KLK3 (PSA), AR (androgen receptor), FOLH1 (PSMA) expression was quantified relative to ACTB (beta actin) using the comparative CT method.

2 × 10⁶ cells per cell line were harvested, pelleted and frozen prior to the assay. RNA was extracted using the RNeasy Mini Kit (250) (Qiagen, Hilden, Germany) and cDNA was generated using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), both according to the manufacturers’ instructions. PRAME specific primers were obtained from Qiagen and DNA amplification was performed using the RT2 SYBR Green fluor qPCR Mastermix (Qiagen, Hilden, Germany) and the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). All data was obtained in triplicate and the mRNA expression levels were analyzed and normalized to HTB78, BLCLs and BT474 cells, (a cell lines with very low PRAME expression), and presented as fold change.

Total RNA was isolated from RM PBMCs using Quick-RNA™ MicroPrep kit (Zymoresearch, Irvine CA) according to the manufacturer's instructions. Briefly, 1 μg total RNA was utilized to synthesize complementary DNA as described previously (19 (link)). The reverse transcribed RNA was analyzed on the 7500 Fast Real-Time PCR System (Applied Biosystems, Grand Island NY) with the RT2 SYBR Green Fluor qPCR master-mix (Qiagen, Germantown MD). The macaque specific primers were utilized for monocyte chemoattractant protein (MCP-1), interleukin 6 (IL-6), Interleukin 1β (IL-1β) and Tumor necrosis factor-α (TNF-α) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table 2). Reactions were performed in triplicates and normalization was done utilizing GAPDH to determine the fold change in expression. The specificity of the qRT-PCR was monitored with a non-template control.

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).

RNA concentration and purity were assessed using a NanoDrop 2000C Spectrophotometer (ThermoFisher Scientific). For cDNA synthesis and genomic DNA elimination in RNA samples RT2 Profiler PCR Arrays was used (Qiagen, Valencia, CA). Real Time PCR (RT-PCR) was performed with RT2 SYBR Green Fluor qPCR Mastermix (Qiagen, Valencia, CA) used with predesigned array plates on MyiQ^™ Real-Time PCR Detection System (Bio-Rad). These plates, RT² Profiler^™ PCR Array Rabbit Innate & Adaptive Immune Responses PANZ-052ZA 96-Well Format (Qiagen, Valencia, CA), detect the expression of 84 tested genes, 5 housekeeping genes and other controls. The PCR protocol consisted of one cycle at 95°C (10 minutes) followed by 40 cycles of 95°C (15 sec) and 60°C (1 min) followed by fluorescence data collection (SYBR Green channel) confirmed then by melting curve analysis. Fold changes in gene expression were determined using the C_t method with normalization to ACTA2, ACTB, GAPDH, LDHA, LOC100346936 controls. To calculate mRNA levels, we used the comparative C_t method (^ΔΔC_t method) [19 (link)].