Rt2 profiler pcr array data analysis

Total RNA was extracted with the RNeasy Mini Kit (Qiagen) and cDNA transcribed with the RT² First Strand Kit (Qiagen). Expression of homeobox genes was analysed by real-time PCR (qRT-PCR) using the Homeobox (HOX) Genes RT² Profiler PCR Array (Qiagen) on a Roche LightCycler® 480 System. Real-time PCR data analysis was performed by using the RT² Profiler™ PCR Array Data Analysis (Qiagen) and based on the ΔΔC_T method with normalization of the raw data to the housekeeping gene RPLP0 after analysis with the BestKeeper software [24 (link)].

The lipid metabolism-related transcriptome was evaluated by real-time PCR using the ready-to-use rat Fatty Acid Metabolism RT² Profiler PCR Array (PARN-007Z, QIAGEN) which profiles the expression of 84 key gene transcripts involved in lipid metabolism. Each cDNA sample was diluted with nuclease-free water and mixed with the RT² SYBR green Mastermix (QIAGEN). Twenty-five μL of the experimental mixture was added to each well of the rat Fatty Acid Metabolism array (one array for each cDNA). Real-time PCR was performed on the CFX96 thermocycler (Bio-Rad, Milan, Italy) using the following cycling conditions: (1) 95°C/10 min and (2) 40 cycles: 95°C/15 sec followed by 60°C/1 min. Dissociation curves were then performed to verify PCR specificity using the default melting curve program of the instrument. Data were analyzed using the QIAGEN RT² Profiler PCR Array Data Analysis Web Portal. RAGE gene expression was carried out according to the manufacturer's instructions using specific rat primers (PPR44508A, QIAGEN) and the same mastermix, thermocycler, and conditions previously indicated. Each sample was run in triplicate, and the fold difference between groups was evaluated by the ΔΔCt method.

RNA from purified human/murine islets/beta-cell line was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA), reverse-transcribed using Super Script II Reverse Transcriptase (Invitrogen) and qRT-PCR analysis was performed using TaqMan assays (Life Technologies) according to the manufacturer’s instructions. Normalized expression values were determined using the ΔCt method. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) data were normalized for the expression of ACTB, and ∆∆Ct (fold change) or ∆Ct values were calculated. Statistical analysis compared gene expression across all cell populations for each patient via one-way ANOVA followed by a Bonferroni post-test for multiple comparisons between the population of interest and all other populations. Statistical analysis was performed also using the software RT² profiler PCR Array Data Analysis (Qiagen). For comparison between two groups, a Student’s t test was employed. Analysis was performed in triplicates before/after 72 h of culture. Reported on Supplementary Table 8 are the characteristics of primers used for human and murine genes.