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Standard microarray protocols include DNA extraction, DNA amplification and labeling, hybridization, washing, scanning, and data analysis.
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Microarray protocols, including labeling, hybridization and post-hybridization washing procedures, were performed as recommended by the manufacturer and are available at https://www.home.agilent.com.agilent.com
Of note, our microarray protocol included an RNA amplification step, which was excluded in the k-PCR analysis.
For microarray, RNA was isolated using the Qiagen RNeasy kit following the manufacturer's protocol, including the addition of DNase.
RNA isolation and microarray protocols were carried out as described in Hu et al [30].
The protocol included 3×12 reps.
The technology use transcript specific color coded molecular probe pairs to capture and count individual transcripts and has several advantages in comparison to microarray platform including easy protocols, less hands-on time and higher sample throughput [ 17].
All protocols related to this microarray platform, which include information on probe sequences and synthesis, labeling, hybridization and scan protocols, and microarray data have been submitted to NCBI's Gene Expression Omnibus database at http://www.ncbi.nlm.nih.gov/geo/ with GEO accession number GSE42597.
The RNA preparation was performed as described for the microarray protocol, without RNA purification.
The microarray protocol follows previously published methods [ 82, 83].
The one-colour microarray protocol is described in manual: G4140-90040.
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