G2505c microarray scanner

Four subpopulations from the luminal MAS9806 xenograft were sorted by FACS based on the presence of EpCAM, SSEA-4 and CD24 cell surface markers on the epithelial MAS9806 cells. Total RNA was isolated from four biological replicates of each population (totally 16 samples). 50–100 ng of total RNA was amplified and labeled with cy3-CTP following Agilent Low Input Quick Amplification Labeling Kit protocol for One-Color Microarray-Based Gene Expression Analysis. Hybridization was performed according to the manufacturer's protocol (Agilent One-Color Microarray-Based Gene Expression Analysis v6.0) using 1650 ng cy3-labeled cRNA per sample and hybridized onto Whole Human Genome Oligo Microarrays (4x44K, G4112F).
The microarrays were scanned using Agilent Technologies Microarray Scanner (G2505C). Data were extracted from the scanned images using Feature Extraction Software (Agilent Technologies), version 10.7 and protocol GE1-107-Sep09 for mRNA, using default settings and FULL text output. One sample from the double positive cell fraction was removed from the further analysis due to poor data quality. Raw data were uploaded to Gene expression omnibus (GEO) accession number GSE48384.

Total RNA from 34 snap frozen xenograft tissue samples was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Total RNA concentration was measured using NanoDrop (NanoDrop Technologies, Wilmington, DE, USA) and the quality was evaluated using 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). A total of 100 to 125 ng RNA was amplified and labeled with cy3-CTP following the Agilent Low Input Quick Amplification Labeling Kit protocol for One-Color Microarray-Based Gene Expression Analysis. Hybridization was performed according to the manufacturer’s protocol (Agilent One-Color Microarray-Based Gene Expression Analysis v6.5) using 600 ng cy3-labeled cRNA per sample and SurePrint G3 Human Gene Expression 8x60K Microarrays. The microarrays were scanned using Agilent Technologies Microarray Scanner (G2505C). Data were extracted from the scanned images using Feature Extraction software (Agilent Technologies), version 10.7 and protocol GE1-107-Sep09 for mRNA. Four xenograft samples (HBCx-11, HBCx-23, HBCx-29, and HBCx-33) were excluded from further analyses due to poor data quality.

RNA molecules were isolated using MirVana Total RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the instructions of the manufacturer. The microarray and the data analysis were performed by Welgene Biotech (Taipei, Taiwan) using the SurePrint G3 GE 8 × 60 K Microarray, 8 × 60 K, AMADID 028005 (Agilent Technologies, USA [24 (link)]). The arrays were scanned with G2505C Microarray Scanner (Agilent). The information of probes on the arrays was extracted from the image data using Feature extraction 10.5.1.1 (Agilent) for quantifying signal and the intensity of background.

Microarray analysis was performed as previously described (Piprek et al. 2018 (link)). Total RNA was labeled with fluorescent dyes using Agilent One-Color Quick Amp Labeling Protocol. RNA isolated from ZW gonads were labeled with Cy3, and RNA from ZZ gonads with Cy5. Fluorescently labeled RNA samples were mixed with Agilent Hi-RPM Hybridization Buffer, and hybridized at 65 °C for 17 h in HybArray12 hybridization station (Perkin Elmer). RNA from ZW and ZZ were mixed together and hybridized to the same chip. The RNA isolated from the gonads in different stages of development was labeled with the same fluorochrome (either Cy3 or Cy5) and hybridized individually to the separate chips. Samples were washed in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; at RT) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; at RT) for 1 min each and immersed in a solution of acetonitrile. Air-dried slides (custom-commercial Agilent-070330 X. laevis Microarray slides) were scanned in the Agilent Technologies G2505C Microarray Scanner at a 5-μm resolution. The microarray experiment was repeated three times.