Part 1: Total RNA Isolation
1. Tumors are surgically removed from patients and rapidly transported to pathology where a pathologist removes a piece(s) for microarray analysis. These tumor samples are rapidly frozen in Liquid Nitrogen and stored at —80 C until use.
2. The frozen tumor specimen is removed from the freezer and, using a scalpel, small pieces (approximately 50-100 milligrams each) are cut off of the larger tumor specimen while it is still frozen. As the small pieces are cut off, they are immediately placed into 10-12mls of
3. TRIzol Reagent (GibcoBRL Life Technologies) that is contained within a 50ml Screw Cap Tube at RT. For a given tumor sample, upto 1 gram can be placed into this volume of TRIzol, and typically 0.5-1 gram of tissue is used per isolation if possible. Note, that as little as 0.2 gram has been successfully used in this volume but it may be better to use 5-8mls TRIzol for specimens of this size (the TRIzol Reagent Protocol calls for 1ml TRIzol per 50-100mg tissue processed).
4. The tumor sample in TRIzol is homogenized using a PowerGen 125 Tissue Homogenizer (Fisher Scientific) starting at 5000 RPM and gradually going up to approximately 20,000 RPM over a period of 30-60 seconds at RT. The homogenization is performed until a homogeneous solution is obtained and very few visible tumor pieces can be seen.
5. Incubate at RT for 5-10 minutes after homogenization.
6. The TRIzol/tumor homogenate is transferred to a 50ml Oak Ridge Centrifuge tube and centrifuged at 12,000g (10,000RPM on SS34) for 5-10 minutes at 4 C.
7. Remove the upper fat layer by using a Pasteur pipette hooked up to a vacuum flask (the upper fat layer, if present, has a yellowish appearance while the TRIzol homogenate remains red).