The same RNA samples were used in microarray hybridizations and real-time PCR reactions.
However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations.
DNase (Qiagen, Valencia, CA, USA) treated total RNA from all biological replicates previously used in microarray hybridizations (4 replicates per condition) were examined using RT-qPCR (see supplemental text for detailed methods).
Transcript expression levels were examined using DNase (Qiagen, Valencia, CA) treated total RNA from each of the three biological replicates used in microarray hybridizations (see Additional file 1 for methods).
Prior to use in microarray hybridizations, RNA samples were quality controlled using a Thermo Scientific NanoDrop™ 1000 Spectrophotometer to assess the quantity and then quality analysed on an Agilent RNA 6000 Nano chip (lab-on-a-chip), using a 2100 Agilent Bioanalyser.
The labeled RNAs were then precipitated with ethanol for use in microarray hybridization.
One μg of RNA was amplified using the amino Allyl MessageAmp ™ aRNA Kit (Ambion, 2130 Woodward St, Austin TX, 78744) to generate amino allyl modified aRNA for use in microarray hybridization.
We modeled secondary structure in the single-stranded target, modeling the target both as DNA and as RNA, at a range of temperatures which is inclusive of hybridization temperatures commonly used in microarray protocols: 37°C, 42°C, 52°C and 65°C.
Given that it was impractical to collect sufficient mRNA from hybrids for direct use in microarray hybridizations, all mRNA samples were then amplified twice using the MessageAmp II aRNA kit (Ambion, Texas, USA).
Four micrograms of mixed labeled aRNA was used in for microarray hybridization.
