Genechip wt pico reagent kit

We carried out the Affymetrix Whole Transcript Expression array process according to the manufacturer's protocol (GeneChip WT Pico Reagent Kit). We synthesized cDNA using the GeneChip WT Pico Amplification kit as described by the manufacturer. The sense cDNA was fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Human 2.0 ST Array at 45°C for 16 hours. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GeneChip™ Command Console software.

RNA (2 ng) isolated from laser-assisted microdissected tissue (somatosensory as well as motor cortex derived from four control and four d10 microglia-depleted mice) was transcribed, amplified and labeled using the GeneChip WT Pico Reagent Kit (Affymetrix) according to the manufacturer’s instructions and subsequently hybridized to a GeneChip® Mouse Gene 2.1 ST 16-Array Plate (Affymetrix) according to the manufacturer’s protocol. For microarray processing (washing, hybridization, signal detection), a GeneTitan® instrument (Affymetrix) was used. The raw data were processed by the Affymetrix Gene Chip Command Console (AGCC) and normalization was performed using the Affymetrix Expression Console (EC). Additionally, quality control was done with the EC by calculating several metrics (AUC, PM-mean, BG-mean, MAD, RLE, spike-ins). After quantile normalization, the data were exported to CHP files, which can be read by the Affymetrix Transcriptome Analysis Console (TAC).

PBMC from 7 untreated RRMS patients [5 females (71.4%); mean age, 39.0 years (8.0); mean disease duration, 7.0 years (5.1)] were plated into 24-well plates for 24 h in the presence or absence of an EZH2 inhibitor (histone deacetylase inhibitor suberoylanilide hydroxamic acid—SAHA) at 1 μg/μl concentration. After 24 h, cells were harvested and total RNA isolated using the RNeasy kit (Quiagen) and hybridized to Affymetrix Human Transcriptome Arrays (HTA 2.0) (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol (GeneChip WT Pico Reagent Kit (Affymetrix)).

After two days with the treatments in liquid medium, the worms were collected and cleaned with M9 buffer 10 times to ensure the elimination of E. coli. RNAi extraction was performed with TRIzol^® following the protocol of PureLink^TM RNA Mini Kit from Invitrogen (Carlsbad, CA, USA).
GeneChip WT Pico Reagent kit from Affymetrix was used to synthetized Ss-cDNA from 3.5 ng of each sample. The following steps of hybridization to GeneChip^®C. elegans Gene 1.1 ST Array Strip (Affymetrix, Santa Clara, CA, USA) with 26 unique sequences of each transcript were performed under the instruction of the manufacturer’s protocol. After scanning, the microarray data were processed using Affymetrix Transcriptome Analysis Console. Three independent RNA samples were employed. The control JK1466 strain and that treated with L-tryptophan-benzyl ester-betaxanthin or L-tryptophan methyl ester-betaxanthin were analyzed. The N2 strain was also analyzed and compared with the JK1466 strain.

For each sample, 1 ng of total RNA was amplified using the GeneChip® WT Pico Reagent Kit (Affymetrix) according to the manufacturer's protocol. 20 μg of cRNA was used as input for the second cycle of cDNA reaction. 5.5 μg of single-stranded cDNA was used as input for the fragmentation reaction. The Affymetrix Genechip WT Terminal labeling kit was used for fragmentation and biotin labeling. Finally, the samples were hybridized to the whole-transcriptome Rat Gene 2.0 ST microarrays (Affymetrix) on the Genechip Fluidics Station 450 (Affymetrix), scanned using the Genechip Scanner 7G (Affymetrix) and the raw intensity values stored in CEL files by the GeneChip® Operating Software (Affymetrix). These raw CEL files were normalized using the Affymetrix® Expression Console Software (version 4.0, Affymetrix) and the adjusted intensity values were transformed to log2 format. The complete microarray dataset is available at Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE110675.

High through-put RNA expression analysis was performed with the Affymetrix GeneChip Human Transcriptome Array 2.0^®, which contains a panel of 70,523 RNA transcripts. Five nanogram total RNA from each individual sample was processed with the Affymetrix GeneChip WT Pico Reagent Kit^® to obtain 5,5 µg ss-cDNA, which was fragmented, labelled and hybridized to the GeneChip^® according to the manufacturer’s instructions. The technical equipment for chip processing was Gene Chip Hybridization Oven 645, Gene Chip Scanner and Gene Chip Fluidics Station 450 Dx (Affymetrix^®, Thermo Fisher Scientific^®, Waltham, MA, USA). Microarray data were processed using the RMA method [27 (link),28 (link)] from the oligo package [29 (link)].