Microarray Probe ID, primer sequences, amplicon sizes, annealing temperature and qPCR efficiency are shown.
QPCR primers were either chosen from previous publications from our laboratory [ 22, 25] (for IRF1, IRF7, IRF10, SACS, Deltex3, STAT1 and ZNFX1) or designed based on sequences representing informative microarray probes using Primer 3 (http://frodo.wi.mit.edu) (for DHX58, IκBα, IL-8, IRAK4, ISG15, PKR, RSAD2, SCYA123, TLR3, TLR9 and ATPS).
The ERCC Informatics subgroup will determine the "correct" sequences to be used for microarray probe and QRT-PCR primer design.
Due to the requirement of designing qPCR primers across exon/introns boundaries, microarray probes and qPCR primer locations varied.
Gene-specific primers different from the primers used for microarray probe generation were used in duplicate PCR reactions (Bio-Rad iQ SYBR Green Supermix) on a Bio-Rad MyCycler.
For IGF1, alternative splicing is unlikely to be the reason behind the lack of correlation, but unfortunately, for almost all other genes (11 out of 15), the real-time PCR primers and microarray probe locations are not in the same exon.
This may be explained by the inability of our microarray probes and PCR primers to discriminate between IRE-positive and IRE negative transcripts.
This is useful information, for example, when designing microarray probes or PCR primers that may fail if they include chimeric sequence.
The locations of real-time PCR probes and primers and of microarray probe sets are shown for the examples of IGF1 and EDNRA in Figure 3.
Primers were designed for short and specific amplification of the microarray probe region of the selected sequences with Primer 3 plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (Additional file 10).
