Rt2 profiler pcr array kit

Total RNA was isolated from adipose tissues stored in RNAlater RNA stabilization reagent (QIAGEN) using TRIZOL (Invitrogen) according to the manufacturer’s instructions. The extracted RNA was reversed transcribed using a similar method as described above for qRT-PCR analysis of fat tissue leptin and adiponectin genes. Expression of genes involved in lipid and cholesterol metabolism was analyzed by PCR array using a 96-well Human Lipoprotein Signaling & Cholesterol Metabolism RT2 Profiler PCR Array Kit (PAMM-080ZC-2, SABiosciences, Valencia, CA, USA) and a LightCycler 480 PCR system (Roche, Basel, Switzerland) according to the kit manufacturer's instructions. The data were analyzed using SDS Software 2.3 and web-based programs at www.SABiosciences.com/pcrarraydataanalysis.php (SABiosciences). Gene expression was normalized to the mean of all house-keeping genes in the array. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were determined using STRING version 10 [21 (link)].

The autophagy -focused gene expression analysis was carried out according to the protocol provided by Qiagen (RT² profiler PCR array kit, PAHS-084Z). Briefly, total cellular RNA was extracted from Huh7 cells treated with or without MPA for 24 h using SuperArray RT² qPCR-Grade RNA Isolation Kit. The elimination of genomic DNA contamination and first strand cDNA synthesis were carried out using RT² First Strand Kit. Autophagy-focused gene expression array was performed using RT² Profiler TM PCR Array. Statistical analysis of data was performed with the Data-Analysis-Template provided by Qiagen.

After desired treatments of HepG2 cells, total cellular RNA was extracted and reverse-transcribed using a similar method as described above for quantitative real-time PCR. Expression of genes involved in lipid metabolism and cholesterol was studied by PCR array using a 96-well Human Lipoprotein Signaling & Cholesterol Metabolism RT²Profiler PCR Array Kit (Qiagen, Hilden, Germany) and LightCycler 480 PCR system (Roche, Basel, Switzerland) according to the kit manufacturer’s protocol.

Total RNA was isolated from Ishikawa and MFE 296 cells grown as monolayer and 3D colonies, using RNeasy Mini kit (Qiagen) following manufacturer's instructions. 500 ng of total RNA was used for the cDNA synthesis using RT² First Strand Kit (Qiagen). Quantitative real-time PCR (Q-PCR) was performed using RT² SYBR Green ROX qPCR Mastermix and RT² Profiler PCR Array kit (Qiagen) on 7900 HT FAST Thermocycler (Applied Biosystems). Amplification and analysis were performed as per manufacturer's instructions. Relative quantification [comparative Ct (ΔΔCt) method] was used to compare the expression level of the test genes with the internal control (arithmetic mean of five housekeeping genes included in the array). Fold change expression of genes in 3D (+ECM) and 2D culture were plotted in log scale range.

RNA was extracted from control HOVEC and MSC CM treated HOVEC by TRIzol method. 500μL of TRIzol (Thermo, Waltham, MA, USA) was used for 200,000 HOVEC cells. The RNA was purified according to manufacturer’s protocol. After RNA purification, 500ng of RNA was used for cDNA conversion. Expression of angiogenesis markers is analyzed with RT2 profiler PCR array kit (QIAGEN, Hilden, Germany) and CFX96 PCR instrument (Bio-Rad, Hercules, CA, USA). Reaction volume per each well was 25μL, which containing 4ng of cDNA and 12.5μLof Universal SYBR green Supermix (Bio-Rad, Hercules, CA, USA). PCR was performed with cycle condition described in manufacturer’s protocol. Gene expression levels are analyzed by the delta-delta CT method.

Aqueous humor RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, United States) and purified by an RNeasy MinElute Cleanup Kit (Qiagen, United States). Complementary DNA (cDNA) was synthesized with a SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, United States). The PCR array for cytokines and chemokines was conducted with an RT² Profiler PCR Array Kit (Qiagen, 330231 PAHS-150ZA, United States) according to the manufacturers’ instructions. A total of 90 cytokines or chemokines were quantified in each sample.