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Gene expression

What happens if the hybridization time is extended beyond 16 hours?

The standard gene expression hybridization time is 14-16 hours at 45°C. At high temperatures and longer incubation times the sample will evaporate. Loss of sample is undesirable for several reasons:

  • Low volume of hybridization solution in the probe array can lead to dry spots that will show up as uneven hybridization and thus, compromise data.
  • Sample loss compromises the possibility of repeating the experiment with the identical sample.
  • Sample evaporation can lead to changes in the salt concentration of the solution which can affect the stringency conditions for hybridization.
I have observed on occasion that multiple _at probe sets are mapped to the same gene but give different expression results. How do I reconcile the difference?

There are various reasons why this happens. With increasing knowledge of the genome, the unique probe sets (_at probe sets) that were initially designed may turn out to represent subclusters that have collapsed into a single cluster in a later design. Therefore, it may seem that multiple "unique" _at probe sets now correspond to a single gene. Different results from the probe sets could be observed due to the following reasons:

  • They represent splice variants or may cross-hybridize to different members that belong to a highly similar gene family or transcripts with different poly-A sites.
  • One probe set is more 5' than the other.
  • One probe set is better designed than the other.

In these cases, it is important to use the resources available on the NetAffx™ Analysis Center to understand if any of the above scenarios apply. Other expression analysis techniques may also be used to confirm which probe set reflects the transcript level more accurately.

Can results from different laboratories and different times be compared with each other directly and how do you control the variables in this type of experiment?

Array results can potentially be compared directly. However, it is important to check the following important elements before doing so:

  • Experimental design strategy should be the same at various sites.
  • Identical target labeling protocols should be followed, and yields from cDNA and IVT reactions should be within the same range as specified for that study.
  • Same algorithm parameters are used.
  • Similar results from 3'/5' ratios, background, noise, and scaling factors. Check arrays for scratches and even hybridization/staining.
  • Comparability of results obtained from different operators should be evaluated before including their results in the same study.

For Research Use Only. Not for use in diagnostic procedures.