RNA extraction protocol

Abdulmawjood et al. (2005) have further refined the use of RT-PCR and demonstrated that use of a specific RNA extraction protocol, coupled to the targeting of a specific region of the GFAP transcript that appears be relatively stable, ensures that the template can be extracted from processed samples in sufficient amounts and quality. In addition, they have also transferred the assay to a real-time PCR platform (see Chapter 7) to enable a quantitative tissue-specific assay to be designed and performed. 

Nucleic acid extraction protocols using guanidine hydrochloride, sodium sarco-syl, and ethanol have been developed to quantify viral RNA by bDNA in lymph node tissue, liver tissue, and peripheral blood monocytes (Wilber and Urdea, 1995). 

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... 

DEVELOPMENT OF HEAT-INDUCED PROTOCOL FOR RNA EXTRACTION FROM FFPE TISSUE... 

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... 

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. 

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. 

Protocol for RNA Extraction from FFPE Tissue Sections... 

Prior to total RNA extraction, sample lysis procedures have to be performed. Lysis conditions are very important for the success of the RNA extraction and depend strongly upon the sample used. Due to great diversity, the biological sample can be pulverized, homogenized, sonicated, or otherwise disrupted to yield a mixture that contains cells, subcellular components, and other biological debris in an aqueous buffer or suspension. Here is described the protocol for the Trizol method of RNA extraction. 

A protocol combining commercial kits for RNA extraction (Amplicor HIV Monitor test, Roche, 073 0246) and one-tube RT-PCR (Titan One-Tube RT PCR system, Boehringer Mannheim, 1 855 476) was established. The procedure described herein is less sensitive than the one described in Subheadings 3.1.1.-3.1.3., because positive amplification results were obtained only starting from 3000 copies/mL. The following protocol was used ... 

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