Heating protocol

There are a number of various commercial products designed for DNA/ RNA extraction, but their use may lead to variable results. Based on the evidence that formalin-induced modification of protein is similar to that of nucleic acid modification by formalin (Fig. 3.1),15-19 our research group at the University of Southern California has conducted a serial study of AR based heating protocols for DNA/RNA extraction from FFPE tissues following our experience of the AR principle as applied to IHC on tissue sections heating under the influence of pH.19-21... 

TABLE 3.1 Comparison of CGH Scores of DNA Samples Extracted from FFPE Tissue Sections between Heating and without Heating Protocols... 

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 heat-induced retrieval protocol for extraction of formaldehyde-modified DNA from FFPE tissue sections provides a simple and effective method of DNA extraction from archival tissue samples.25,45 Based on PCR using three primer pairs ranging from 152-541 bp and a real time KTC-PCR analysis, the heat-induced retrieval protocol yields a better quality and quantity of DNA samples extracted from FFPE tissue sections than conventional methods of extraction.24 In addition, this heating protocol may provide an alternative approach for DNA extraction in some cases such as a recent publication by Ferrari et al. mentioned above.34... 

Following the development of successful heating protocols for DNA extraction from archival FFPE tissue sections as described, it required no great leap of imagination to evaluate similar methods for RNA extraction. Analysis of... 

Based on the heat-induced AR principle, DNA/RNA extraction from FFPE tissues can be successfully achieved by a simple heating protocol that allows satisfactory application of molecular analysis using FFPE tissue samples housed in pathology laboratories worldwide. By a combination of improved extraction methods with various innovative techniques of molecular biology, more reliable results of molecular profiling for archival tissue are anticipated. 

Based on the similarity of formalin-induced chemical modification between nucleic acids and proteins, the efficiency of heating protocols for DNA/RNA extraction has been demonstrated (see Chapter 3 for detail). Basic AR principle including heating condition and pH value of AR solution as well as certain chemicals may play roles to establish optimal protocols. 

Aryl-5,6-dihydrobenzo[4,5]imidazo[l,2-C]-quinazolines Isatoic anhydride could also be reacted with amino-, hydroxyl- or thiolanilines to form 2-(2-aminophenyl)benz-imidazoles, oxazoles or thiazoles, Scheme 5.38. In the case of 2-(2-aminophenyl)benzimidazoles (X=N), the product was formed after 3 min at 150°C in acetic acid. The products could subsequently be further elaborated 6-aryl-5,6-dihydrobenzo[4,5]imidazo[l,2-C]quinazolines, a four ring system, was formed by treatment of the 2-(2-aminophenyl) benzimidazole (X=N) with different aldehydes in acetic acid at 150°C for 5 min (J. Westman, and K. Orrling, Personal Chemistry, Uppsala, Sweden, unpublished results). Fifteen compounds were synthesised in 20-75% overall yield. This 3 + 5 min procedure should be compared to the conventional heating protocol developed by Devi and co-workers57, where each reaction step was run overnight to eventually afford the products in only 30-50% yield. 

Figure 7.8 Observed regioisomeric isomers of the N-arylated benzimidazole product obtained from the microwave-heated protocol.

The acid addition and heating protocol were designed to avoid excessive foaming and boiling within the microwave-digestion vessel. 

The Friedlander annulation is one of the most straightforward approaches towards poly-substituted quinolines. Thus, a 22-membered library of quinolines was synthesized in a TsOH-catalyzed cyclocondensation-dehydration of 2-aminoaryl ketones and 2-aminoarylaldehydes with ketones in a household microwave oven (with power control) under solvent-free conditions [112]. It was observed that the Friedlander reaction occurred readily also in an oil-bath (at 100 °C). To compare the conventional and dielectric heating conditions precisely, a purpose-built monomode microwave system with temperature control was employed instead of the household oven. The experiments at 100 °C under otherwise identical conditions demonstrated that the dielectric heating protocol was only slightly faster. Products were isolated by a simple precipitation-neutralization sequence (in the case of solid products) or neutralization-extraction for oily or low melting point products (Scheme 43). 

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