Heating protocols hybridization

Figure 3.3 Comparison of array CGH among DNA extracted from fresh tissue, FFPE tissue by heating protocol or nonheating protocol for two human tissue samples of metastatic carcinoma in lymph node (a-c), and undifferentiated non-small cell carcinoma (d-f). Array CGH hybridization genomic profiles show ratio values representing relative copy number of single BACs. A good result is scored as 1.0 that indicates a low standard deviation for gains (>0.2), normal (0.0), or losses (<-0.2). In these two cases, fresh samples show best score as 2, both FFPE tissue samples show identical score of 3. Each spot represents the average of three replicates. Clones are ordered by chromosomal position as numbers at the bottom (x axis) of each picture. The y axis is the log2 ratio of test reference intensity. Provided by Sandy DeVries from Dr. Frederic Waldman s Lab at UCSF.

The role of high temperatures in the breakdown of protein crosslinks introduced by aldehydes has been discussed earlier in this book. The aforementioned combined technique is especially effective in increasing the in situ hybridization signal in archival tissues, the fixation history of which may or may not be known. This protocol also detects low levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme digestion alone. 

The incubation of the elution buffer with the Dynabeads can be done on a heat block (as written in the Panomics protocol) or in a water bath as well. The use of the hybridization oven is also convenient if it is not being used to hybridize the membranes at the time. 

Samples such as Drosophila embryos or egg chambers are easily manipulated in suspension in 0.5-ml PCR tubes (Protocol 2.8), in which solutions are changed by allowing the tissue to settle to the bottom of the tube, aspirating the liquid carefully, and replacing with 400-500 xl of the new solution. This is by far the most convenient way to perform the hybridization and requires a minimum volume for all washing steps. The denaturation step is also easy to perform consistently, because the sample can be heated and annealed in a thermal cycler or conventional water bath. 

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