Bioarray highyield rna transcript labeling kit

Transcriptome profiling was performed using Affymetrix Gene Chip RaGene 2.0 st v1 arrays (Santa Clara, CA, USA), according to the instructions of the manufacturer. Three independent biological replicates were processed per condition, resulting in a total of 24 samples. The quality and quantity of RNA were assessed using a Bioanalyzer (model 2100 with RNA 6000 Nano Chips, Agilent Technologies, Amstelveen, The Netherlands). Two hundred Nano gram of total RNA from each sample was used to prepare double-stranded cDNA, from which biotin-labeled cRNA was synthesized using the BioArray HighYield RNA Transcript Labeling Kit (Enzo). After purification on a QIAGEN RNeasy column, the cRNA was fragmented and hybridized to the arrays. Hybridization, washing and scanning of the arrays were performed according to the manufacturer's instructions. The image data were processed using Affymetrix MAS 5.0 software to generate gene expression data, which were normalized using a robust multi array (RMA) protocol (Bolstad et al., 2003 (link)) and then assessed for statistical significance using a 3-way ANOVA test implemented in the Partek Genomic Suite (http://www.partek.com).

Hybridizations were carried out as described by Lisowska et al., [29 (link)]. Briefly, double-stranded cDNA was synthesized with Reverse Transcriptase II (Life Technologies, Carlsbad, CA, USA) using 8 μg of total RNA as a template. Obtained cDNA (16 μg) was then used for the synthesis of biotinylated cRNA with the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY, USA). Both cDNA and cRNA were purified with GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA, USA). Fragmented cRNA was hybridized first to the Affymetrix control Test3 Array, and then, after evaluating the samples’ quality, to the Human Genome U133 Plus 2.0 Array (Affymetrix) for 16 h at 45°C. The microarrays were stained, washed, and subsequently scanned with GeneChip Scanner 3000 (Affymetrix). Data were acquired using the GCOS 1.2 software (Affymetrix).

Biotin-labeled cRNA was prepared by in vitro transcription using BioArray High Yield RNA Transcript Labeling Kit (ENZO Life Sciences, Inc., Farmingdale, NY) following the standard Affymetrix protocols. Biotinylated cRNAs were cleanup, quantified and 15 μg were fragmented and added to a hybridization cocktail according to Affymetrix procedures.
After quality determination on Test-3 arrays, the samples were hybridized using the GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA) for 16 h at 45 °C to GeneChip Human Genome U133 set (HG-U133A and HG-U133B). The arrays were washed and stained with streptavidin–phycoerythrin using the Affymetrix antibody amplification protocol for eukaryotic targets (EukGE-WS2 protocol) in the GeneChip Fluidics Station 400 (Affymetrix, Santa Clara, CA). Images were acquired using the GeneArray Scanner (Agilent Technologies, Palo, CA), and row data were collected and analyzed by using the Affymetrix Micro Array Suite (MAS) version 5.0 software.

Evaluation of the changes in the expression profile of pyroptosis-related genes in the analyzed cells was carried out using the oligonucleotide microarray method. For this purpose, the HG-U133A 2.0 plate set (Affymetrix, Santa Clara, CA, USA) was used in accordance with the manufacturer’s recommendations.
In order to prepare the matrix for the analysis in the first step, cDNA synthesis was carried out with the SuperScript^® Choice System kit (Invitrogen Life Technologies, Waltham, MA, USA). In turn, the BioArray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences, Inc, Farmingdale, NY, USA) was used to obtain biotinylated cRNA. Next, the labeled cRNA were subjected to a fragmentation step using the Sample Cleanup Module kit (Qiagen GmbH, Hilden, Germany). This process was carried out for 35 min at 94 °C. Hybridization of the cRNA to HG-U133A microarrays labeled with a phycoerythrin-streptavidin complex was the final step of the analysis. The scanning was performed using a GeneArray Scanner G2500A (Agilent Technologies, Inc., Santa Clara, CA, USA).

Expression microarray analysis was performed by a GeneChip Human Genome U133 Plus 2.0 expression microarray (Affymetrix, Santa Clara, CA, USA). From 8 μg of total RNA, first-strand cDNA was synthesized with SuperScript III reverse transcriptase (Invitrogen) and T7-(dT)24 primer (Amersham Biosciences, Little Chalfont, UK). Double-stranded cDNA was then synthesized, and biotin-labeled cRNA was synthesized using a Bio-Array HighYield RNA transcript-labeling kit (Enzo Life Sciences, Farmingdale, NY, USA). Twenty micrograms of labeled cRNA was fragmented and hybridized to the GeneChip oligonucleotide microarray with a GeneChip hybridization control kit. The microarray was stained and scanned according to the Affymetrix protocol. The scanned data were processed using GeneChip operating software 1.4. The signal intensity of each probe was normalized so that the average signal intensity of all the probes on a microarray would be 500. The average signal intensity of all the probes for a gene was used as its transcription level. Genes were classified into those with high (>1000), moderate (250–1000), or low (<250) transcriptions according to their signal intensities [30 (link)].

hTERT-OA 13A FLSs and hTERT-RA 516 FLSs were plated at 85–90% confluence in 100 mm plates. The next day, DMEM containing 10% FBS was added. Forty-eight hour later, total RNA was extracted using Trizol reagent (Qiagen, Valencia, CA) and subjected to microarray analysis. Briefly, double stranded DNA was synthesized using SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA). The DNA product was purified using GeneChip sample cleanup module (Affymetrix, Santa Clara, CA). cRNA was synthesized and biotin labeled using BioArray high yield RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY). The cRNA product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The fragmented, biotinylated cRNA was hybridized to HG-U133_Plus_2 gene chip using Affymetrix Fluidics Station 400. Fluorescent signal was quantified during two scans using Agilent Gene Array Scanner G2500A (Agilent Technologies, Palo Alto, CA) and GeneChip operating Software (Affymetrix). Genesifter software (VizX Labs, Seattle, WA) was used for the analysis of fold changes.