Alexa fluor 555 reactive dye

Microarrays using the isolated RNA were performed by the RCUG team of Hannover Medical School and as previously described in detail in Schwarzer et al. Briefly, when possible, 100 ng (or less if not available) of total RNA was used to prepare Aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp II Kit; Thermo Fisher Scientific), applying one round of amplification as directed by the company, except for a 2-fold downscaling of all reaction volumes. Before the reverse transcription reaction, 1 μL of 1:5,000 dilution of Agilent’s One-Color Spike-in Kit stock solution (Agilent Technologies) was added to the total RNA used for each sample. The labeling of aaUTP-cRNA was performed with Alexa Fluor 555 Reactive Dye (Thermo Fisher Scientific) following the manufacturer’s instructions with the Amino Allyl MessageAmp II Kit (2-fold downscaled reaction volumes). Afterward, cRNA fragmentation, hybridization, and washing steps were carried out as recommended in “One-Color Microarray-Based Gene Expression Analysis Protocol V5.7,” except that 500 ng of each fluorescently labeled cRNA population was used for hybridization. Slides were scanned using the Agilent Micro Array Scanner G2565CA (pixel resolution 3 μm, bit depth 20). Data extraction was performed with the “FeatureExtraction Software V10.7.3.1” using the extraction protocol file “GE1_107_Sep09.xml.”