Wt plus reagent kit

Collected RNA samples were subjected to microarray assay to determine a gene expression profile. We utilized Affymetrix Clariom D microarray chips for profiling. The RNA sample were first prepared using the WT PLUS reagent kit (Affymetrix, Thermo Fisher Scientific, Waltham, MA, USA) followed by hybridization on the Clariom D microarray chips. The raw data of Clariom D chips were first subjected to quality control examination pursuant to the Affymetrix manuals. The chips that passed the quality control criteria were then analyzed with Partek; a commercial software specific for microarray data analysis.

As previous reports have described [23 (link)], total mRNA samples from 18 healthy controls, 18 febrile controls, and 18 KD patients 24 h before IVIG was given (KD1 group), and 18 KD patients 21 days after IVIG therapy (KD2 group) were pooled together into three RNA libraries, each containing RNA samples from 6 patients. All RNA samples were then prepared for hybridization to the GeneChip^® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara) using the WT PLUS Reagent kit. Hybridized HTA 2.0 microarray chips were checked for quality, and the gene expression data was then analyzed with commercially available software (Partek, St. Louis, MI, USA).

RNA spleen samples were prepared for whole-transcriptome expression analysis using the WT PLUS Reagent Kit following the manufacturer’s recommended protocol (Affymetrix, Inc.). Next, 100 ng total RNA was used to prepare the hybridization-ready targets. Individual sense-strand DNA targets were randomized and hybridized to Mouse Gene 2.1 ST 96-Array Plates (Affymetrix, Inc.) using the GeneTitan Multi-Channel Instrument for hybridization, staining, and washing of arrays, as well as for scanning. Quality control (QC) metrics for hybridization, labeling and sample quality were generated using the Affymetrix Expression Console (version 1.3.187) software. All samples passed QC criteria.

The preparation of total RNA from hearts was performed hearts using the RNeasy Midi Kit (Qiagen, Hilden, Germany). The extraction procedure was conducted in accordance with the manufacturer’s manual. RNA integrity was checked with RNA Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples in this study showed high quality RNA Integrity Numbers (RIN; mean 8.9). RNA was further analyzed by photometric Nanodrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies).
Synthesis of biotin labeled cDNA was conducted on three replicates of both experimental groups according to the manufacturers´ protocol (WT Plus Reagent Kit; Affymetrix, Inc). Briefly, 200 ng of total RNA were converted to cDNA. After amplification by in vitro transcription and another cDNS synthesis cycle, cDNA was fragmented and biotin labeled by terminal transferase. Finally, cDNA was hybridized to Affymetrix Mouse Transcriptome 1.0 arrays, washed and stained and scanned on the GC Scanner 3000 with G7 update as described in the standard Affymetrix GeneChip protocol (Version 2). Signal summarization using the RMA algorithm as well as statistical analysis were performed using the Affymetrix TAC software. The significance threshold was set to p < 0.05 and 0.01.

RNA extracts were prepared using the Affymetrix WT PLUS Reagent Kit (Affymetrix). RNA quality and quantity were assessed using a 2100 Agilent Bioanalyzer (Agilent) and a NanoDrop instrument (Thermo Scientific), respectively. For gene expression analysis, 100 ng of total RNA was used in conjunction with the Affymetrix standard protocol for Human Transcriptome Arrays 2.0 (Affymetrix Inc.). For the miRNAs analysis, 1 μg of total RNA was analysed using the FlashTag Biotin HSR RNA labelling kit for the Affymetrix Genechips miRNA 4.0 microarrays (Affymetrix Inc.).

Gene expression arrays were performed to compare gene expression differences between the control and pH 6.4 and DCA-exposed groups in CP-A. After extraction of RNA, RNA concentration and integrity were assessed with microfluidic analysis using the Agilent 2100 Bioanalyzer and denaturing agarose gel electrophoresis. Labeling and hybridization were performed as follows: 100 ng of total RNA was amplified and labeled using the WT Plus reagent Kit (Affymetrix) and then hybridized to Clariom S human (Affymetrix, USA). Washing and scanning were conducted using the GeneChip System of Affymetrix (GeneChip Hybridization Oven 645, Gene-Chip Fluidics Station 450, GeneChip Scanner 3000 7G). A TA threshold of FC ≤ -2 for downregulated genes and FC ≥ 2 for upregulated genes was used. The list of genes was analyzed using Transcriptome Analysis Console (TAC). A series of processes and microarray analyses were performed at GeneticLab Co. Ltd. (Hokkaido, Japan).