Biomark 96.96 dynamic array

Quantitative RT-PCR was performed essentially as described (39 (link)) using RNA isolated from hippocampus (constitutive Nrxn2 KOs and Nrxn2 nKOs). RNA was isolated using TRIzol reagent according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Isolated RNAs (1 ng) were subjected to target-specific RT and 16 cycles of PCR preamplification with forward and reverse primers of the detection assays. Preamplified cDNAs were then processed for real-time PCR analysis on the Biomark 96:96 Dynamic Array according to the manufacturer’s protocol (Fluidigm, South San Francisco, CA, USA). Fluorescein (FAM) dye–coupled detection assays were purchased from Integrated DNA Technologies (Coralville, IO, USA): All probes were used as previously described (25 (link)); please see table S1 for detailed information. To ensure the specificity of the amplification, all assays were tested with dilutions of mouse hippocampal cDNA to verify high efficiency (90 to 110%) and linear amplification [coefficient of determination (R²) > 0.96]. Transcript levels were normalized to the internal control β-actin.

Total RNA isolated from the 48 chickens was reverse-transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) following manufacturer’s instructions. cDNAs was diluted 1:20 and subjected to a Specific Target Amplification step using PreAmp Master Mix kit (Fluidigm Corporation) with a mixture of all primer pairs and 14 cycles of pre-amplification. The BioMark™ 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously measure the expression of selected genes using Real-Time PCR Analysis User Guide PN 68000088 K1. Primers used for qPCR reactions are listed in Additional file 3. Data were analyzed using HTqPCR R package [15 (link)] and normalized considering GAPDH, RPS8 and TOP2B as reference genes, as suggested after GeNOrm analysis [16 (link)].

Gene expression levels were analyzed using qPCR in micro-Fluidigm. The qPCR EvaGreen experiment was performed on a Biomark 96.96 dynamic array (Fluidigm, San Fransisco, California, USA). Among the samples and primers assessed with the chip, 4 pairs of primers (Table 1) were tested against 12 samples, corresponding to the three distinct times of culture (8h, 16h, 24h) for three biological replicates with a technical replicate for each sampling time. An internal control of human DNA was used to check efficiency and quality of the run. For each cDNA sample, targeted cDNA was amplified using the pool of primers and TaqMan^® PreAmp Master Mix (Fluidigm, San Francisco, California, USA) with the following program: (i) 10 minutes at 95°C, (ii) 14 cycles of 15 seconds at 95°C and 4 minutes at 60°C. The samples were then treated with exonuclease Exo I (for 30 minutes at 37°C for digestion and for 15 minutes at 80°C for inactivation). Finally, samples were added to a pre-mix (2X TaqMan Gene Expression Master Mix, 20X DNA Binding Dye Sample Loading Reagent, 20X EvaGreen^® and TE buffer) before being loaded into the macro-array. The sets of primers were loaded into the macro-array at a concentration of 20 μM. Control idnT values were used to normalize data.

The extent of expression of human IFN–stimulated genes was measured as described previously [11 (link)]. High-throughput qPCR was performed with BioMark 96 × 96 Dynamic Array (Fluidigm Corporation) according to manufacturer’s protocol. cDNAs (50ng/μl) were pre-amplified with all the primers and analyzed with the BioMark real-time PCR instrument. Initial data analysis was performed with the fluidigm real-time PCR analysis software. Hierarchical clustering was performed in Partek with Pearson dissimilarity and complete linkage.

Gene expression was analyzed using the BioMark 96·96 Dynamic Array (Fluidigm). Data were analyzed using BioMark Real-Time PCR Analysis Software Version 3.0.2 (Fluidigm). The TaqMan Gene Expression Assay Mixes used in this study are listed below.

High-throughput real-time quantitative PCR was performed on a BioMark 96.96 Dynamic Array (Fluidigm Corp., San Francisco, CA, USA) with TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA, USA) at the Weizmann Institute of Science (Rehovot, Israel). Three biological replicates were used for each treatment and two technical replicates were analysed for each biological replicate. Primers (Supplementary Table S3) were designed with Primer3 software, and synthesized by Metabion (Steinkirchen, Germany) and Hylabs (Rehovot, Israel). Expression levels of the target genes were normalized to the control gene actin.