Probe synthesis, hybridization conditions, and wash regimen were performed as described above for hybridization reactions.
This conclusion was supported by experimental observations in our laboratories indicating that probes derived from rat and mouse cDNAs yield inconsistent results in Northern blotting under stringent hybridization conditions (data not shown).
Furthermore, the predictions are likely to be affected by the hybridization conditions.
Because SNP alleles only differ in one nucleotide and because it is difficult to achieve optimal hybridization conditions for all probes on the array, the target DNA has the potential to hybridize to mismatched probes.
Isolation of nucleic acids, gel electrophoresis, hybridization conditions, oligonucleotide synthesis and standard in vitro recombinant techniques were carried out as described elsewhere (27, 28).
In this report, we show solution-phase hybridization behavior on a three-dimensional microarray surface [ 10 ] and demonstrate that hybridization conditions, specifically the time allowed for hybridization, can impact the accuracy of fold-change calculations.
Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization.
No single hybridization condition is optimal for all oligonucleotide probes, so it is inevitable that there is variability among the signals within a probe set.
Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.
When we applied the algorithms to ten experiments done with either human brain or human lung replicate samples (same mRNA, slightly different hybridization conditions), the results were all similar.