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Gene expression

For microarray analysis, what happens if the hybridization time is extended beyond 16 hours?

The standard gene expression hybridization time is 16 hours at 45 degrees C, rotating at 60 rpm. 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 compromise the 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?

Microarray probes are designed to bind to different regions of a gene's transcripts. The "_at" suffix in Affymetrix probe set IDs represents a unique transcript cluster, indicating that all probes in that set should hybridize to a unique gene. However, it's not uncommon for multiple probe sets to map to the same gene, especially for genes that have multiple transcript variants.

Differences in expression results from different probe sets for the same gene could be due to several reasons. For example, the gene may produce different mRNA transcripts through alternative splicing, resulting in some probe sets binding to exons that are included in some transcript variants, but not others. In addition, not all probes are equally efficient at hybridizing to their targets. Some might bind more strongly or specifically than others, leading to differences in signal intensity.

How can a mismatch probe have a higher intensity than its complementary perfect match probe?

There can be a number of reasons for this. It is possible that the probe sequence has high homology with another unknown sequence, resulting in a high Mismatch-to-Perfect Match ratio. Another possibility is that the probe sequence on the array is correct for the majority of the cases, however, the sample may have a sequence variation that causes low specific binding to the Perfect Match, and high specific binding to the Mismatch.

Regardless of the cause, the redundancy of the number of probes representing a sequence on GeneChip expression arrays negates any significant impact this may have on the final interpretation of the data.

For Thermo Fisher Scientific microarrays, what does high background mean?

A high background implies that impurities, such as cell debris and salts, are binding to the probe array in a nonspecific manner and that these substances are fluorescing at 570 nm (the scanning wavelength). This nonspecific binding causes a low signal to noise ratio (SNR), meaning that genes for transcripts present at very low levels in the sample may incorrectly be called Absent. High background creates an overall loss of sensitivity in the experiment.

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