Nonheating protocols

We performed a study to apply the a-CGH technique to test the quality of DNA extracted from FFPE tissues by different methods, using a nonheating protocol, a heat-induced extraction protocol, based on AR as applied to IHC, and comparing the findings to extracts from paired fresh frozen tissue samples (unpublished data). The study was conducted in two stages. 

First, a limited study was performed using breast cancer tissue to establish an optimal protocol of DNA extraction for a-CGH analysis that would allow comparison of a-CGH results after boiling in different solutions three pH values of 7, 9, and 12 of Britton and Robinson buffer solution, and a 0.1 M sodium hydroxide solution. DNA samples extracted from frozen and from FFPE tissue sections by a nonheating protocol were employed, and the results were compared a protocol of boiling samples in 0.1 M sodium hydroxide gave optimal results. 

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.

Combining both heating and nonheating protocols employed in a sequential order were evaluated, but without any advantage (Fig. 3.4). RT-PCR was performed by standard methods, RNA extracted from fresh MDA cells and human tissue of breast cancer with known tested genes was used as positive control, and pure water was used to replace template (cDNA) as negative control for every experiment of PCR. To assure the accuracy of PCR tests, all reactions were performed in triplicate. 

Notes. MDA cell line fixed for variable times h, hour D, day H, heating S, nonheating protocol Positive, positive control. All terms of genes are comparable with Figure 3.4, all figures under each gene are the size of band (bp). Black column (I), showing band of PCR product with correlated bp (see Fig. 3.4). Empty column, no band. 

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