One color quick amp labeling protocol

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.