《英语教学》（英文版）8 RNA Purification

peratures cannot be overemphasized. In addition, yields from very small amounts of starting material are subject to the law of dimin ishing returns. Thus, if the option is available, always choose more starting material rather than less Samples can be pooled together, if possible, to maximize yields. For example 5 mg of tissue or 2.5 x 10 cells yields about 10 of total RNA, comprised of 8ug rRNA, 0.3 ug mRNA, 1.7 ug tRNA, and other RNA. In comparison, 1 g of tissue or 5 x 10 cells yields about 2mg of total RNA, comprised of 1.6mg rRNA 60ug mRNA +333 ug tRNA and other rna Is There Protein in Your RNA Preparation, and If So, Should You be concerned? Pure RNA has an A260: A20 absorbance ratio of 2.0. However for most applications, a low A260: A2so ratio probably wont affect the results, Researchers at ambion Inc have used total rna with A260 2s0 ratios ranging from 1. 4 to 1. 8 with good results in RNase protection assays, Northern analysis, in vitro translation experi ments, and RT-PCR assays. If protein contamination is suspe to be causing problems, additional organic extractions with an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1 mixture)may remove the contaminant Residual phenol can also lower the A260: A2so ratio, and inhibit downstream enzymatic reac tions Chloroform/isoamyl alcohol (24: 1)extraction will remove residual phenol. Chapter 4, " How to Properly Use And Maintain Laboratory Equipment, discusses other artifacts that raise and lower the A2602s0 ratio Some tissues will consistently produce RnA with a lower A260-2s0 ratio than others the A260-2s0 ratio for rna iso- lated from liver and kidney tissue, for example, is rarely above 1.7. Is Your RNa Physically Intact? Does It Matter? The integrity of your rNa is best determined by electrophore sis on a formaldehyde agarose gel under denaturing conditions. The samples can be visualized by adding 10 ug/ml of Ethidium Bromide(EtBr)(final concentration) to the sample before load ng on the gel. Compare your prep's 28S rRNa band (located at approximately 5 Kb in most mammalian cells) to the 18S rRNA band (located at approximately 2.0 Kb in most mammalian cells In high-quality rna the 28S band should be approximately twice the intensity of the 18S band(Figure 8.1). The most sensitive test of Rna integrity is northern analy sing a high molecular weight probe expressed at low levels in the sues being analyzed. However, this method of quality control is very time-consuming and is not necessary in most cases 202 Martin et al

peratures cannot be overemphasized. In addition, yields from very small amounts of starting material are subject to the law of dimin￾ishing returns. Thus, if the option is available, always choose more starting material rather than less. Samples can be pooled together, if possible, to maximize yields. For example, 5 mg of tissue or 2.5 ¥ 106 cells yields about 10mg of total RNA, comprised of 8mg rRNA, 0.3mg mRNA, 1.7mg tRNA, and other RNA. In comparison, 1 g of tissue or 5 ¥ 108 cells yields about 2 mg of total RNA, comprised of 1.6mg rRNA + 60mg mRNA + 333mg tRNA and other RNA. Is There Protein in Your RNA Preparation, and If So, Should You Be Concerned? Pure RNA has an A260 :A280 absorbance ratio of 2.0. However, for most applications, a low A260 :A280 ratio probably won’t affect the results. Researchers at Ambion, Inc. have used total RNA with A260:280 ratios ranging from 1.4 to 1.8 with good results in RNase protection assays, Northern analysis, in vitro translation experi￾ments, and RT-PCR assays. If protein contamination is suspected to be causing problems, additional organic extractions with an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1 mixture) may remove the contaminant. Residual phenol can also lower the A260 :A280 ratio, and inhibit downstream enzymatic reac￾tions. Chloroform/isoamyl alcohol (24:1) extraction will remove residual phenol. Chapter 4, “How to Properly Use And Maintain Laboratory Equipment,” discusses other artifacts that raise and lower the A260:280 ratio. Some tissues will consistently produce RNA with a lower A260:280 ratio than others; the A260:280 ratio for RNA iso￾lated from liver and kidney tissue, for example, is rarely above 1.7. Is Your RNA Physically Intact? Does It Matter? The integrity of your RNA is best determined by electrophore￾sis on a formaldehyde agarose gel under denaturing conditions. The samples can be visualized by adding 10mg/ml of Ethidium Bromide (EtBr) (final concentration) to the sample before load￾ing on the gel. Compare your prep’s 28S rRNA band (located at approximately 5Kb in most mammalian cells) to the 18S rRNA band (located at approximately 2.0Kb in most mammalian cells). In high-quality RNA the 28S band should be approximately twice the intensity of the 18S band (Figure 8.1). The most sensitive test of RNA integrity is Northern analysis using a high molecular weight probe expressed at low levels in the tissues being analyzed. However, this method of quality control is very time-consuming and is not necessary in most cases. 202 Martin et al

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 203

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 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

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 procedures follow Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly. The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion since rna is more tube after 12 to 24 hours of centrifugation at 232, 000rpul? Of th dense than DNA and most proteins, it pellets at the bott This classic method yields the highest-quality RNA of any avail- able technique. Small RNAs(e. g, 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered(Mehra 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of amples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999)with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974) Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some what cumbersome if large sample numbers are to be processed The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours(although somewhat cum bersome) without resorting to ultracentrifugation. RNA mole cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single step method( Chomczynski and Sacchi, 1987) is based on the propensity of RNa molecules to remain dissolved in the aqueous phase in a solution containing 4 M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the Martin et al

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 procedures follow. Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly.The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion. Since RNA is more dense than DNA and most proteins, it pellets at the bottom of the tube after 12 to 24 hours of centrifugation at ≥32,000rpm. This classic method yields the highest-quality RNA of any avail￾able technique. Small RNAs (e.g., 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered (Mehra, 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of samples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999) with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974). Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some￾what cumbersome if large sample numbers are to be processed. The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours (although somewhat cum￾bersome) without resorting to ultracentrifugation. RNA mole￾cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single￾step method (Chomczynski and Sacchi, 1987) is based on the propensity of RNA molecules to remain dissolved in the aqueous phase in a solution containing 4M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the interphase. 204 Martin et al

It is not difficult to find researchers who swear by GITC- phenol procedures because good-quality RNA, free from geno- mic dNa contamination is quickly produced. However, a se- cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997 S. Herzer, personal communication). There is no single expla nation for these polarized opinions, but the following should be considered Problems can occur in the procedure during the phenol/chloro form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1 For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings The interface between the aqueous and organic layers is another potential source of genomic contamination. To get higl purity RNA, avoid the white interface(can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you'll probably want as much of the aqueous layer as possible e. again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessiv salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed br washing the RNa pellet with 70% EtoH(ACS grade). To the RNA pellet, add about 0.3 ml of room temperature (or -20oC 70% ethanol per 1.5ml tube or approximately 2 to 3 ml per 15 to 40ml tube Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 x g; approximately SS34 rotor), for 5 to 10 minutes at room temperature or ayos 7500rpm in a microcentrifuge, or approximately 5500rpm in Table 8. Spin Requirements for Phenol Chloroform Extractions Tube Speed Spin Time 1.5ml 10,000×g 2.0ml 15 ml 12,000×g 50ml 12,000×g RNA Purification 205

It is not difficult to find researchers who swear by GITC— phenol procedures because good-quality RNA, free from geno￾mic DNA contamination is quickly produced. However, a se￾cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997; S. Herzer, personal communication). There is no single expla￾nation for these polarized opinions, but the following should be considered. Problems can occur in the procedure during the phenol/chloro￾form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1. For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings. The interface between the aqueous and organic layers is another potential source of genomic contamination. To get high￾purity RNA, avoid the white interface (can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you’ll probably want as much of the aqueous layer as possible, again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessive salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed by washing the RNA pellet with 70% EtOH (ACS grade). To the RNA pellet, add about 0.3ml of room temperature (or -20°C) 70% ethanol per 1.5ml tube or approximately 2 to 3ml per 15 to 40 ml tube. Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 ¥ g; approximately 7500 rpm in a microcentrifuge, or approximately 5500 rpm in a SS34 rotor), for 5 to 10 minutes at room temperature or at 4°C. RNA Purification 205 Table 8.1 Spin Requirements for Phenol Chloroform Extractions Volume Tube Speed Spin Time 1.5 ml 10,000 ¥ g 5 minutes 2.0 ml 12,000 ¥ g 5 minutes 15 ml 12,000¥ g 15 minutes 50 ml 12,000¥ g 15 minutes

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large Non-Phenol-Based methods Very fast, clean RNA, can process large sample numbers, pos ble genomic contamination One major drawback to using the guanidium acid-phenol nethod is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro- pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium- based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin DNA and proteins are washed off, and the rna is eluted at the end in an aqueous buffe This technique yields total Rna of the same quality as he phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods(Ambion, Inc unpublished observations). Since these are column-based proto- cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication(on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e, high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems A rea- sonable starting condition for the clarifying spin is 8 minutes at 7650xg. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et al

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ethanol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large. Non-Phenol-Based Methods Very fast, clean RNA, can process large sample numbers, possi￾ble genomic contamination. One major drawback to using the guanidium acid-phenol method is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro￾pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium-based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin. DNA and proteins are washed off, and the RNA is eluted at the end in an aqueous buffer. This technique yields total RNA of the same quality as the phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods (Ambion, Inc., unpublished observations). Since these are column-based proto￾cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour. The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication (on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e., high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems. A rea￾sonable starting condition for the clarifying spin is 8 minutes at 7650 ¥ g. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular￾weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et al