G2539a dual laser scanner

1 mm sized as homogeneous as possible pieces were isolated from the lateral walls of LA, RA, LV and RV, not close to the SA or AV node, or the bundle of His or origin of its branches in the interventricular septum. Total RNA was immediately extracted in separate vials with RNAEasy Minikit (Qiagen, Germantown, MD, USA), following manufacturer's instructions. RNA concentration was determined before and after reverse transcription in the presence of Cy3/Cy5 dUTP with a WU-83060-00 Thermo Scientific NanoDrop ND-1000 and its quality with a 2100 Bioanalyzer (Agilent, DE). 825 ng of differently (Cy3/Cy5) labeled biological replicas of the same chamber were hybridized 17 h at 65 °C with GPL10333 Agilent-026655 Whole Mouse Genome Microarray 4 × 44 K v2. The chips were washed and scanned with an Agilent G2539A dual laser scanner at 5 μm resolution in 20-bit scan mode and primary analysis performed with (Agilent) Feature Extraction 11.6 software.
Agilent microarrays and Illumina NextSeq 500 (equally available to us) provide similar expression ratios⁸⁴ . However, in addition to being considerably cheaper, microarrays were preferred because their open protocol allowed optimization to significantly reduce the technical noise. More importantly, microarray raw data are of reasonable size to allow the extensive COR analysis with the available computer resources.

We have used our standard protocol [36 (link)] for RNA extraction, purification, reverse transcription and fluorescent labeling, and hybridization with Agilent human 4 × 44 k gene expression two-color G2519F microarrays. (Full experimental details can be found in the genomic datasets cited at Section 3.1 below, deposited by us in https://www.ncbi.nlm.nih.gov/gds/?term=iacobas). The chips were scanned with an Agilent G2539A dual laser scanner and primary analysis performed with (Agilent) feature extraction v.12.0 software. All Agilent products (equipment, consumables and software were purchased from Agilent Technologies, Santa Clara, CA 95051, USA (https://www.agilent.com/cs/agilent/en/contact-us/united-states) All control spots as well as spots with corrupted or saturated pixels, or with forward fluorescence less than twice the background one in any of the four profiled biological replicas were removed from the analysis of that type of samples.

Lungs were quickly removed, frozen in liquid nitrogen, and stored at −80 °C. Our optimized protocol [37 (link),38 ] and the “multiple yellow” strategy [39 (link)] were used to profile each “condition” in four biological replicas. Briefly. total RNA was extracted with Qiagen RNeasy minikit, concentration was determined with a NanoDrop ND-2000 Spectrophotometer, and purity determined with an Agilent RNA 6000 Nano kit in an Agilent 2100 Bioanalyzer. Total RNA was then reverse-transcribed in the presence of Cy3/Cy5 dUTP and the incorporation of fluorescent tags was determined again with the nanodrop. Differently labeled samples of biological replicates were hybridized overnight with Agilent 60 mer 4 × 44k whole genome rat V2 arrays (#G2519F). The arrays were scanned with an Agilent G2539A dual-laser scanner, and primary analysis was performed with (Agilent) Feature Extraction 11.1 software. All spots affected by local corruption or with foreground fluorescence less than twice the background were disregarded, and data were normalized and filtered following our standard procedure [40 (link)].