1234567891011 Figure 8.1 Assessing qual- ity of rNa preparation via 9.5 (A) This gel shows total RNA samples (5ug/lane) ranging from high-quality, intact RNa (lane 2)to almost 4.4 totally degraded RNA (lane 7). Note that as the rNA is 2.4 degraded, the 28S and 18S ribosomal bands become less distinct, the intensity of the 1.35- ribosomal bands relative to the background staining in the lane is reduced. and there is a significant shift in their apparent size as compared size standards. (B)This autorad of the same gel hybridization with a biotinylated GAPDH RNA probe followed by noniso- topic detection. The exposur is 10 minutes the day after the chemiluminescent sub. strate was applied. Note that the signal in lane 2, from intact RNA. is well local- 点二三 ing below the bands, or when the RNa is extremely de- graded, no bands at all (lane f Ambion. In Northern analysis is not tolerant of partially degraded RNA 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 North ern blot by 20%. Nuclease protection assays and RT-PCR analy ses 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 s &y n a chaotropic agent such as guani- thods of isolating total RNA from cells and tissue samples. mos dium or a detergent to break open the cells and simultaneously RNA Purification 203Northern 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 North￾ern blot by 20%. Nuclease protection assays and RT-PCR analy￾ses 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 9.5 – 7.5 – 4.4 – 2.4 – 1.35 – .24 – 1 2 3 4 5 6 7 8 9 10 11 Figure 8.1 Assessing qual￾ity of RNA preparation via agarose gel electrophoresis (A) This gel shows total RNA samples (5mg/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 noniso￾topic detection. The exposure is 10 minutes the day after the chemiluminescent sub￾strate was applied. Note that the signal in lane 2, from intact RNA, is well local￾ized with minimal smearing, whereas the signals from degraded RNA samples show progressively more smear￾ing below the bands, or when the RNA is extremely de￾graded, no bands at all (lane 7). Reprinted by permission of Ambion, Inc. A B