Rt2 profiler pcr array system

A panel of genes mined from our microarray result was validated using a real-time quantitative PCR (qPCR) assay as described previously [28 (link)]. The selection of these genes was justified by a brief literature survey (Table 1); those genes, which showed involvement with the functions identified in the present study (Results and Discussion of present project) and past findings (listed in the tables), were selected for validation. The primers of the selected genes were available in catalogued assays and were purchased as a custom array plate from SAB Biosciences (Fredrick, MD). qPCR was carried out using the RT² Profiler PCR Array System (SA Biosciences, MD) on the BioRad CFX system (BioRad Laboratories, Inc., Hercules, CA) following manufacturers’ instructions. Two housekeeping genes (ACTB and GAPDH) were used for normalization. Data was analyzed using the Excel-based RT² profiler PCR Array Data Analysis using the 2^∆∆ct method (Fig 4). qPCR outputs were compared to the array results. We considered these values mutually validated if the outcomes from the two assay platforms showed regulation in same direction, i.e., either both up- or down-regulated with -fold change greater than ±1.5.

Total RNA sample was isolated using an RNeasy Minikit protocol (Qiagen, Hilden, Germany) from the liver of saline or AdIGF-I-MSC-treated mice 1 day after systemic administration. Equal amounts of RNA from different samples were then pooled and reverse-transcribed to cDNA. The quality of the cDNA conversion was confirmed by PCR on pooled samples, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Expression levels of 84 genes involved in the cell cycle were evaluated using the RT2 profiler PCR array system (catalog number: PAMM-020Z; SABiosciences, Frederick, MD, USA) following the manufacturer’s instructions. The mRNA expression levels obtained for each gene were normalized to the expression of the GAPDH housekeeping gene. Finally, data results were analyzed by the manufacturer’s online website and scores were expressed as fold change versus saline group (fold change: ΔΔCt).

Frozen samples of the distal colon from 6 mice per group, about 45 mg in size, were homogenized using pistils. RNA was extracted using the Illustra RNAspin Mini RNA isolation Kit (GE Healthcare, Buckinghamsire, UK) according to the manufacturer’s instructions. After RNA extraction, cDNA was synthesized from 1µg of total RNA using the RT2 First Strand Kit (SABioscience, Frederick, MD, USA) as described by the manufacturer. The cDNA was then used to detect the expression level of genes involved in Wnt signaling by qPCR using the RT2 Profiler PCR array system specific for the Wnt/β-catenin signaling pathway (Ref.: PAMM-043Z, SA Biosciences). SYBR Green Master Mix (SA Biosciences) was prepared and used according to the manufacturer’s instructions. Briefly, a master mix containing 1µg of cDNA was prepared, and 25 µL of the mix was added to each well of the PCR array. The qPCR was performed in a Chromo 4 thermal cycler (MJ Research, Bio-Rad, Hercules, CA, USA) under the following conditions: 1 cycle of 10 min at 95 °C, to activate the HotStart DNA polymerase, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. SYBR Green fluorescence was measured at every cycle.