It is an absolute necessity to use controls together with the transgenic sections. The most obvious negative control is to apply the probe specific for the transgene to sections from nontransgenic animals. Numerous artifacts may be apparent. When initial criteria for nonartifactual signals have been met, it can be further confirmed with some of the following:
1. The signal is not present if the tissue is digested with RNase A prior to hybridization.
2. The signal can be decreased or eliminated by competing the hot label with a large (10-20-fold) excess of cold label.
3. The signal is changed in intensity by known physiological changes
4. The use of a randomer oligonucleotide of the same length does not produce a similar signal.
If possible, a positive control should also be run. Use sections from a tissue known to contain the mRNA of interest.
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