The Agilent One-Color Microarray Hybridization protocol (Agilent Technologies) was utilized for microarray hybridizations following manufacturer's recommendations.
Microarray hybridizations were conducted utilizing the Agilent QuickAmp, One-Color Microarray Hybridization protocol (Agilent Technologies) following manufacturer's instructions.
Microarray hybridization was performed in Gene-Machines hybridization chamber (Gene-Machines, San Carlos, CA, USA) following by washing according to Agilent oligonucleotide microarray hybridization protocol.
Genomic DNA was extracted as above and fed into the established barcode PCR and microarray hybridization protocol (Pierce et al. 2007; Hoon et al. 2008).
Fish experiments, design of the rainbow trout cDNA microarray, hybridization protocol and data analyses are described in detail elsewhere [ 10, 11].
Thus, two different protocols were used to generate the chemostat compendium's samples for microarray hybridization: Protocol A and Protocol B. The main difference between these protocols is that Protocol A includes the polyA-mRNA isolation step (with cDNA synthesis being performed on purified mRNA), while Protocol B excludes the purification step (with cDNA synthesis being performed on total RNA).
This is mainly because the data often has been generated with different microarray platforms, hybridization protocols, and the authors use different methods and different thresholds to calculate differentially expressed genes (DEGs) [8].
For microarray hybridization, the protocol described in the Affymetrix GeneChip eukaryotic one-cycle target preparation protocol, using 5 μg of total RNA, was followed.
Microarray processing and hybridization protocols have been described previously [ 36, 45].
Four μg of labelled RNA were fragmented to a uniform size and hybridized to the custom array following the Combimatrix CustomArray 90K Microarray Hybridization and Imaging Protocol Arrays were stripped and re-hybridised using the CustomArray Stripping Kit for 90K (CombiMatrix Cat. No. 610049) following the protocols of the manufacturer.
