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Molecular Biology Problem Solver 22

Molecular Biology Problem Solver 22. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | A 9.5- 7.5- 4.4- 2.4- 1.35- .24- B Figure 8.1 Assessing quality of RNA preparation via agarose gel electrophoresis A This gel shows total RNA samples 5 mg lane ranging from high-quality intact RNA lane 2 to almost totally degraded RNA lane 7 . Note that as the RNA is degraded the 28S and 18S ribosomal bands become less distinct the intensity of the ribosomal bands relative to the background staining in the lane is reduced and there is a significant shift in their apparent size as compared to the size standards. B This is an autorad of the same gel after hybridization with a biotinylated GAPDH RNA probe followed by nonisotopic detection. The exposure is 10 minutes the day after the chemiluminescent substrate was applied. Note that the signal in lane 2 from intact RNA is well localized with minimal smearing whereas the signals from degraded RNA samples show progressively more smearing below the bands or when the RNA is extremely degraded no bands at all lane 7 . Reprinted by permission of Ambion Inc. Northern analysis is not tolerant of partially degraded RNA. If samples are even slightly degraded the quality of the data is severely compromised. For example even a single cleavage in 20 of the target molecules will decrease the signal on a Northern blot by 20 . Nuclease protection assays and RT-PCR analyses will tolerate partially degraded RNA without compromising the quantitative nature of the results. Which Total RNA Isolation Technique Is Most Appropriate for Your Research There are three basic methods of isolating total RNA from cells and tissue samples. Most rely on a chaotropic agent such as guani-dium or a detergent to break open the cells and simultaneously RNA Purification 203 inactivate RNases. The lysate is then processed in one of several ways to purify the RNA away from protein genomic DNA and other cellular components. A brief description of each method along with the time and effort involved the quality of RNA obtained and the scalability of the .