RNA samples were harvested from adherent PBMC-derived IL-4 and IL-15 DCs and analyzed using Illumina Human RefSeq-8 Expression BeadChips (Illumina, San Diego, CA, USA). The RNA quantity was determined with the Agilent RNA 6000 Nano Kit and Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). The synthesis of double-stranded cDNA and in vitro transcription were performed with the Ambion Illumina Total Prep Kit (Illumina) according to the manufacturer's instructions. For each sample, input quantity for the first strand synthesis was normalized to 200 ng. After in vitro transcription reaction, yield of purified cRNA was assessed with the RiboGreen assay and the quality was assessed with the Agilent Bioanalyzer. BeadChip hybridization, staining and scanning were performed according to Illumina whole genome expression for BeadStation. For each sample, input of cRNA was normalized to 1500 ng. As a control, Stratagene Universal Human Reference (SUHR) RNA was labeled with the Ambion Total Prep Kit. The labeled cRNA was used as interchip hybridization replicates and showed strong correlation. Biological replicate pairs were analyzed, and for unnormalized data, the linear r2 was >0.94 for all replicates.
Ambion total prep kit
Manufactured by Thermo Fisher Scientific
The Ambion Total Prep Kit is a lab equipment product designed for the extraction and purification of total RNA from a variety of sample types, including cells, tissues, and microorganisms. The kit provides a simple and efficient method for obtaining high-quality RNA for downstream applications such as RT-PCR, Northern blotting, and microarray analysis.
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2 protocols using ambion total prep kit
1
Gene Expression Analysis of IL-4 and IL-15 DCs
2
Illumina Expression BeadChip Microarray Analysis
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Total RNA was processed at the UCLA Neuroscience Genomics Core (UNGC) and quantified using a Ribogreen fluorescent assay. Intersample RNA concentrations were normalized to 10 ng/μL prior to amplification. Amplified and labeled cRNA was produced using the Illumina-specific Ambion TotalPrep kit based on a T7-based linear amplification method 25 . First and second strand cDNA was produced using the Ambion kit and purified using a robotic assisted magnetic capture step. Briefly, this step employs the use of an oligo(dT) primer coupled to the phage T7 RNA polymerase promoter Expression BeadChip (Illumina, San Diego, CA), containing 22,523 probes per array. Hybridization was followed by washing, blocking, staining, and drying on the Little Dipper processor. Array chips were scanned on the Beadarray reader, and expression data was extracted and compiled using BeadStudio software (Illumina).
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