Feature extraction software version 11

Data extractions from Images were performed using the Agilent’s Feature Extraction software version 11. Feature extracted data were analyzed using the DirectArray Version 2.1 software from Agilent. Normalization of the data was performed with DirectArray using the ranked median quantiles according to Bolstad et al. (2003) (link). To identify significantly differentially expressed genes log₂-fold changes are calculated and Student’s t-test was performed. In summary, raw data were normalized by rank median quantiles, intensity values from replicate probes were averaged, log₂-ratios between the treatments were calculated and Student’s t-statistics applied to test for significance. Genes with log₂-fold change <–1 or >1 and p-value < 0.05 were considered to be significantly different. All data show expression levels of genes regulated by P. indica relative to the control levels without P. indica. Differentially expressed genes were then assigned using the A. thaliana Gene Ontology software (TAIR’s GO annotations) (Berardini et al., 2004 (link)) and transcript abundance were classified based on their functional categories and pathways using the MapMan³ software.
The microarray data have been submitted to NCBI (GEO) under the accession number GSE63500.

Data extractions from Images were performed using the Agilent’s Feature Extraction software version 11. Feature extracted data were analyzed using the DirectArray Version 2.1 software from Agilent. Normalization of the data was performed with DirectArray using the ranked median quantiles according to Bolstad et al. [94 (link)]. To identify significantly differentially expressed genes log₂-fold changes are calculated and Student’s t- test was performed. In summary, raw data were normalized by rank median quantiles, intensity values from replicate probes were averaged, log₂-ratios between the treatments were calculated and Student’s t-statistics applied to test for significance. Genes with log₂-fold change < −1 or > 1 and p-value < 0.05 were considered to be significantly different. Genes were classified based on functional categories and pathways using the MapMan (http://mapman.gabipd.org/web/guest/ mapman) and A. thaliana Gene Ontology softwares (TAIR’s GO annotations) [95 (link)].
Microarray data were verified by qRT-PCR as described previously from three independent biological experiments with three technical replicates (Additional file 2: Table S4). The microarray data have been submitted to NCBI (GEO) under the accession number GSE63500 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63500).

TIF files were extracted using Feature Extraction Software version 11 (Agilent Technologies, Santa Clara, CA, USA) and the GE1_1105_Oct12 protocol. The resulting raw data were analysed using DirectArray Software (OakLabs, Hennigsdorf, Germany). The signal distributions of the raw data were visualized using box plots to identify potential issues for individual samples.
Samples were quantile normalized and subjected to statistical analysis (PA versus GA) by applying Welch’s test and calculating log₂ fold changes for each gene. P < 0·050 was considered statistically significant.

About 4 µg of labeled Cy-3-CTP cRNA was fragmented at 60°C for 30 min, and the reaction was stopped by adding 2× GE HI-RPM hybridization buffer (Agilent Technologies, In situ Hybridization kit, Part Number: 5190-0404). The hybridization was carried out in Agilent’s Surehyb Chambers at 65°C for 16 hr. The hybridized slides were washed using Gene Expression Wash Buffer 1 (Agilent Technologies, Part Number: 5188-5325) and Gene Expression Wash Buffer 2 (Agilent Technologies, Part Number: 5188-5326) and were scanned using Agilent Scanner (Agilent Technologies, Part Number: G2600D). Data extraction from the images was done using Feature Extraction Software Version 11.5.1.1 of Agilent.

A total of 62 RNA samples were used for gene expression analysis. To determine gene expression profiles, 4x44K oligonucleotide microarrays (Rat Gene Expression 4x44K v3 Microarray Kit, G2519F-028282, Agilent Technologies) were used. The procedures for hybridization using the fluorescent dye Cy3 followed the manufacturer’s protocols (One-Color Microarray-Based Gene Expression Analysis-Quick Amp Labeling and miRNA Complete Labeling and Hyb Kit, Agilent Technologies). A subset of 58 RNA samples was used for miRNA expression analysis, using the whole rat miRNA 8x15K oligonucleotide microarrays (Rat miRNA Microarray slide, G4471A-070154, Agilent Technologies), containing probes for 758 rat miRNAs based on miRBase database (release 21.0). The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted by Agilent Feature Extraction software version 11.5.1.1 (https://www.agilent.com/) for both gene and miRNA expression. Spots with two or more flags (low intensity, saturation, controls, etc.) were considered as NA, that is, without valid expression value. All microarray raw data is deposited in GEO public database (http://www.ncbi.nlm.nih.gov/geo), a MIAME compliant database, under accession reference Series number GSE229760 for mRNA and miRNA data.

Total RNA was prepared from the A549 cells treated with DMSO alone (control), TGFβ1 (5 ng/ml) and DMSO, TGFβ1 and NCB-0846 (3 µM) or TGFβ1 and NCB-970 (3 µM) using a miRNeasy Mini Kit (Qiagen) and subjected to miRNA expression profiling with the use of an Agilent Human miRNA Microarray kit 8 × 60 K rel.21.0 (Agilent) in accordance with the manufacturer’s protocol. The scanned images were analysed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters to obtain the background subtracted value. The GeneView files were generated using Agilent’s Feature Extraction software version 11.5.1.1. The microarray data were deposited in the NCBIs Gene Expression Omnibus database under the accession number GSE95766 (released on March 08, 2017).

Array − CGH analysis on both patients and their parents was carried out using the SurePrint G3 Custom CGH Microarray, 8 × 60 K 4 × 180 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol version 7.1, using appropriate Agilent Reference DNAs (Euro male and Euro female). The arrays were analyzed with the Agilent Microarray Scanner, Feature Extraction Software version 11.5, and Agilent Genomic Workbench 7.0.