Hybrid probes are known as cDNA or cRNA, because they are complementary to the target molecule. In developing an in situ hybridization protocol, it is vital to learn optimal temperatures and times needed for formation of the hybrid between the cDNA probe or cRNA probe and unique RNA or DNA in the cell. The optimal hybridization temperature depends on several factors, including the types of bases in the target sequence and the concentration of certain ingredients in the media. The concentration of cytosine and guanine in the sequence plays an important role. A cytochemist will use these factors to calculate optimal temperature when planning the experiment. One must be as careful in setting up an in situ hybridization experiment as one is when setting up a test tube hybridization assay. The cytochemist and molecular biologist work together to optimize the conditions.
Another vital consideration in developing good in situ hybridization techniques is the specificity of the probe itself. If the investigators know the exact nucleotide sequence of the mRNA or DNA in the cell, they can design a complementary probe and have it made in a molecular biology lab. However, if the investigators do not know the exact sequence, they may try a sequence that is as close to exact as possible (such as from a related species). For example, they might try a cDNA probe for a mouse DNA sequence on a tissue preparation from a rat. This may or may not work because, if over 5 percent of the base pairs are not complementary, the probe will bind only loosely to the target. This loose binding may cause it to be dislodged in the washing or detection steps and hence the reaction will not be detected, or only some of the sites may be detected and the labeling will not accurately reflect all of the target sites.
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