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To serve as a reference for RNA abundance calibration, a sample must:
- produce detectable signal at each array feature,
- contain all species in the mixture at known concentrations,
- and be easily and accurately reproduced.
Several nucleic acid mixtures satisfy these criteria, including genomic DNA in organisms with low complexity genomes, equimolar mixtures of PCR products or oligos complementary to all array features, and oligos complementary to common sequences present in all array features. Although genomic DNA has been used successfully as a reference sample for E. coli microarray experiments (Wei, et al.), we have not pursued this method because it is unlikely to work with large, high complexity genomes such as humans and and mice.
Calibrated oligo reference: a population of labeled oligos of known abundance that hybridize perfectly to the probe sequences of a microarray. Our system uses either of two types of calibrated oligo references instead of cDNA references, whose transcript abundances are unknown, to obtain absolute abundance information from microarrays. The two kinds:
- Universal oligo reference: an oligo that hybridizes to a common sequence on each microarray feature, e.g. the universal priming sequence of the yeast ORF PCR products (Research Genetics)
- Equimolar oligo reference: an equimolar mixture of different oligos that hybridize perfectly and uniquely to the oligo feature sequences, e.g. the reverse complement sequences of the yeast oligo array (Qiagen Operon)
Like cDNA references, both kinds of calibrated references correct for spot size and shape as well as probe concentration. However, because equimolar oligo references hybridize to the same probe sequence as the target cDNAs, they should also correct for sequence-specific differences in hybridization efficiency, thereby yielding more accurate results. We estimate the retail cost of an equimolar mixture based on the Qiagen Operon set of 6,300 yeast oligos at $9 per hybridization.
Analyses of both types of calibrated oligo references are described further in:
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