《英语教学》（英文版）8 RNA Purification

Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) 8 RNA Purification Lori A. Martin, Tiffany. Smith, Dawn Obermoeller Brian Bruner Martin Kracklauer and Subramanian Dharmaraj Selecting a Purification Strategy Do Your Experiments Require Total RNa or mRNA? 198 Is It possible to predict the total rna yield from a Certain mass of tissue or number of cells? 20 Is There Protein in Your RNA Preparation, and If So, Should You be concerned 202 Is Your RNa Physically Intact? Does It Matter? Which Total RNA Isolation Technique Is Most Appropriate for Your research? What Protocol modifications should Be used for RNA Isolation from difficult tissues? Is a One-Step or Two-Step mRNA(poly (A)RNA)- Purification Strategy Most Appropriate for Your Situation? 209 How Many Rounds of Oligo(dT)-Cellulose Purification Are required? 210 Which Oligo(dT)-Cellulose Format Is Most 210 Can Oligo(dT)-Cellulose Be Regenerated and Reused?.. 211 Can a Kit Designed to Isolate mRNA Directly from he Biological Sample Purify mRNA from Total RNA?... 212 Maximizing the Yield and Quality of an RNA Preparation.. 212 What Constitutes“ RNase- Free Technique”?

197 8 RNA Purification Lori A. Martin, Tiffany J. Smith, Dawn Obermoeller, Brian Bruner, Martin Kracklauer, and Subramanian Dharmaraj Selecting a Purification Strategy . . . . . . . . . . . . . . . .. . . . . . . . . 198 Do Your Experiments Require Total RNA or mRNA? . . . . . 198 Is It Possible to Predict the Total RNA Yield from a Certain Mass of Tissue or Number of Cells? . . . . . . . . 201 Is There Protein in Your RNA Preparation, and If So, Should You Be Concerned? . . . . . . . . . . . . . . . . . . . . 202 Is Your RNA Physically Intact? Does It Matter? . . . . . . . . . . 202 Which Total RNA Isolation Technique Is Most Appropriate for Your Research? . . . . . . . . . . . . . . . . . . . . . 203 What Protocol Modifications Should Be Used for RNA Isolation from Difficult Tissues? . . . . . . . . . . . . . . . . 207 Is a One-Step or Two-Step mRNA-(poly(A) RNA)- Purification Strategy Most Appropriate for Your Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 How Many Rounds of Oligo(dT)–Cellulose Purification Are Required? . . . . . . . . . . . . . . . . . . . . . . . . . 210 Which Oligo(dT)–Cellulose Format Is Most Appropriate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Can Oligo(dT)–Cellulose Be Regenerated and Reused? . . . 211 Can a Kit Designed to Isolate mRNA Directly from the Biological Sample Purify mRNA from Total RNA? . . . 212 Maximizing the Yield and Quality of an RNA Preparation . . . 212 What Constitutes “RNase-Free Technique”? . . . . . . . . . . . . 212 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)

How Does depc inhibit rnase? 2|3 How Are DEPC-Treated Solutions Prepared? Is or Should You Treat Your Pre-made Reagents with r213 More depc better Should You Prepare Reagents with DEPC-Treated Wate DEPC? ..2|4 How Do You Minimize RNA Degradation during Sample Collection and storage ..2|4 How Do You Minimize RNA Degradation during Sample Disruption? Is There a Safe Place to Pause during an rna Purification Procedure? 2|8 What Are the Options to Quantitate Dilute RNA 2|8 What Are the Options for Storage of Purified RNAS 2l9 Troubleshooting 220 A Pellet of Precipitation RNA Is Not Seen at the End of he rna Purification A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance RNA Was Prepared in Large Quantity, but in a downstream Reaction RT PCr is Example My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not observed 222 Only a Fraction of the Original RNA Stored at-70C Remained after Storage for Six Months 222 Bibliography SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly (A)-selected RNa (also commonly referred to as mRNA) This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by Martin et al

How Does DEPC Inhibit RNase? . . . . . . . . . . . . . . . . . . . . . . 213 How Are DEPC-Treated Solutions Prepared? Is More DEPC Better? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Should You Prepare Reagents with DEPC-Treated Water, or Should You Treat Your Pre-made Reagents with DEPC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Collection and Storage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Disruption? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Is There a Safe Place to Pause during an RNA Purification Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options to Quantitate Dilute RNA Solutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options for Storage of Purified RNA? . . . . . . . 219 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet of Precipitation RNA Is Not Seen at the End of the RNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 RNA Was Prepared in Large Quantity, but it Failed in a Downstream Reaction: RT PCR is an Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not Observed . . . . . . . . . . . . . . . . 222 Only a Fraction of the Original RNA Stored at -70°C Remained after Storage for Six Months . . . . . . . . . . . . . . 222 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly(A)-selected RNA (also commonly referred to as mRNA). This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision. From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by 198 Martin et al

RT-PCR. The preference for total RNA reflects the challenge of purifying enough poly (A) RNA for the application (mRNA comprises <5% of cellular RNA), the potential loss of a particu- lar message species during poly(A)purification, and the difficulty uantitating small amounts of purified poly (A)RNA. If the data generated with total Rna do not meet your expectations, using poly() RNa instead might provide the sensitivity and pecificity that your application requires. The pros and cons with either choice are discussed below. Your experimental data will provide the best guidance in deciding whether to use total or poly(A)RNA. Be flexible and open minded; there are many vari- ables to consider when making this decision Two situations where using poly (A)RNA is essential are cDNA library construction, and preparation of labeled CDNA for hybridization to gene arrays. To avoid generating cDNA libraries with large numbers of ribosomal clones, and nonspecific labeled cDNA it is crucial to start with poly(A)RNA for these procedures. The next section gives a brief description of the merits and demerits of using total RNA or poly (A)RNA in some of the most common RNA analysis techniques. Chapter 14, "Nucleic Acid Hybridization, discusses the nuances and quirks of these proce dures in greater depth. For detailed RNa purification protocol see Krieg(1996)Rapley and Manning(1998), and Farrel (1998) Northern Hybridizations Northern analysis is the only technique available that can deter mine the molecular weight of an mRNA species. It is also the least sensitive. Total RNA is most commonly used in this assay, but if you dont detect the desired signal, or if false positive signals from ribosomal RNA are a problem, switching to poly(A)RNA might be a good idea. Since only very small amounts of poly(A)RNA are present, make sure that it is feasible and practical to obtain enough starting cells or tissue. Theoretically you could use as much as 30 ug of poly(A)RNA in a Northern, which is the amount found in approximately 1 mg of total RNA. Will it be practical and feasible for you to sacrifice the cells or tissue required to get this much RNA? If not, use as much poly(A)RNA as is practical One drawback to using poly (A)RNA in Northern hybridiza tions is the absence of the ribosomal rna bands, which are ord of the rna amples, as discussed later in this chapter. Fortunately there are other strategies besides switching to poly(A)RNa that can be used to increase the sensitivity of Northern hybridizations. You could alter the hybridization conditions of the Dna probe RNA Purification 199

RT-PCR. The preference for total RNA reflects the challenge of purifying enough poly(A) RNA for the application (mRNA comprises <5% of cellular RNA), the potential loss of a particu￾lar message species during poly(A) purification, and the difficulty in quantitating small amounts of purified poly(A) RNA. If the data generated with total RNA do not meet your expectations, using poly(A) RNA instead might provide the sensitivity and specificity that your application requires. The pros and cons with either choice are discussed below. Your experimental data will provide the best guidance in deciding whether to use total or poly(A) RNA. Be flexible and open minded; there are many vari￾ables to consider when making this decision. Two situations where using poly(A) RNA is essential are cDNA library construction, and preparation of labeled cDNA for hybridization to gene arrays. To avoid generating cDNA libraries with large numbers of ribosomal clones, and nonspecific labeled cDNA it is crucial to start with poly(A) RNA for these procedures. The next section gives a brief description of the merits and demerits of using total RNA or poly(A) RNA in some of the most common RNA analysis techniques. Chapter 14, “Nucleic Acid Hybridization,” discusses the nuances and quirks of these proce￾dures in greater depth. For detailed RNA purification protocols, see Krieg (1996) Rapley and Manning (1998), and Farrel (1998). Northern Hybridizations Northern analysis is the only technique available that can deter￾mine the molecular weight of an mRNA species. It is also the least sensitive. Total RNA is most commonly used in this assay, but if you don’t detect the desired signal, or if false positive signals from ribosomal RNA are a problem, switching to poly(A) RNA might be a good idea. Since only very small amounts of poly(A) RNA are present, make sure that it is feasible and practical to obtain enough starting cells or tissue.Theoretically you could use as much as 30mg of poly(A) RNA in a Northern, which is the amount found in approximately 1 mg of total RNA. Will it be practical and feasible for you to sacrifice the cells or tissue required to get this much RNA? If not, use as much poly(A) RNA as is practical. One drawback to using poly(A) RNA in Northern hybridiza￾tions is the absence of the ribosomal RNA bands, which are ordi￾narily used to gauge the quality and relative quantity of the RNA samples, as discussed later in this chapter. Fortunately there are other strategies besides switching to poly(A) RNA that can be used to increase the sensitivity of Northern hybridizations. You could alter the hybridization conditions of the DNA probe RNA Purification 199

Anderson, 1999), or you could switch to using RNA probes in the hybridization, which are 3- to 5-fold more sensitive than DNA probes in typical hybridization buffers (Ambion Technical Bulletin 168, and references therein). Dramatic differences in the sensitivity of northern blots can also be seen from using different hybridization buffers. If you remain dissatisfied with the Northern data, and you are not interested in determining the size of the target, switching to a more sensitive technique such as nuclease protection or RT-PCR might help. Nuclease protection assays, which are 5-to 10-fold more sensitive than traditional membrane hybridizations, can accommodate 80 to 100 ug of nucleic acid in a single experiment RT-PCR can detect extremely rare messages, for example, 400 copies of a message in a 1 ug sample as described by Sun et al ( 1998). RT-PCR is currently the most sensitive of the RNa analy- sis techniques, enabling detection and quantitation of the rarest of targets. Quantitative approaches have become increasingly reliable with introduction of internal standards such as in com- petitive PCR strategies(Totzke et aL., 1996; Riedy et al., 1995) Dot slot blots In this procedure, RNA samples are directly applied to a mem- brane, either manually or under vacuum through a filtration manifold. Hybridization of probe to serial dilutions of sample can quickly generate quantitative data about the expression level of a target. Total RNA or poly (A)RNA can be used in this assay. Since the RNa is not size-fractionated on an agarose gel, a potential drawback to using total RNA in dot/slot blots is that signal of interest cannot be distinguished from cross-hybridization t rRNA. Switching to poly(A)RNA as the target source might alle- viate this problem. However. it is crucial that relevant positive and negative controls are run with every dot/slot blot, whether the source of target nucleic acid is total RNA or poly(A)RNA Hybridization to Gene Arrays and Reverse Dot blots Gene arrays consist of cDNA clones(sometimes in the form of PCR products, sometimes as oligonucleotides)or the correspond- ing oligos spotted at high density on a nylon membrane, glass slide, or other solid support. By hybridizing labeled cDNA probes reverse transcribed from mRNA, the expression of potentially hundreds of genes can be simultaneously analyzed. This procedure requires that the labeled cDNa be present in excess of the target spotted on the array. This is difficult to achieve unless poly(a) RNA is used as template in the labeling reaction 200 Martin et al

(Anderson, 1999), or you could switch to using RNA probes in the hybridization, which are 3- to 5-fold more sensitive than DNA probes in typical hybridization buffers (Ambion Technical Bulletin 168, and references therein). Dramatic differences in the sensitivity of Northern blots can also be seen from using different hybridization buffers. If you remain dissatisfied with the Northern data, and you are not interested in determining the size of the target, switching to a more sensitive technique such as nuclease protection or RT-PCR might help. Nuclease protection assays, which are 5- to 10-fold more sensitive than traditional membrane hybridizations, can accommodate 80 to 100mg of nucleic acid in a single experiment. RT-PCR can detect extremely rare messages, for example, 400 copies of a message in a 1mg sample as described by Sun et al. (1998). RT-PCR is currently the most sensitive of the RNA analy￾sis techniques, enabling detection and quantitation of the rarest of targets. Quantitative approaches have become increasingly reliable with introduction of internal standards such as in com￾petitive PCR strategies (Totzke et al., 1996; Riedy et al., 1995). Dot/Slot Blots In this procedure, RNA samples are directly applied to a mem￾brane, either manually or under vacuum through a filtration manifold. Hybridization of probe to serial dilutions of sample can quickly generate quantitative data about the expression level of a target.Total RNA or poly(A) RNA can be used in this assay. Since the RNA is not size-fractionated on an agarose gel, a potential drawback to using total RNA in dot/slot blots is that signal of interest cannot be distinguished from cross-hybridization to rRNA. Switching to poly(A) RNA as the target source might alle￾viate this problem. However, it is crucial that relevant positive and negative controls are run with every dot/slot blot, whether the source of target nucleic acid is total RNA or poly(A) RNA. Hybridization to Gene Arrays and Reverse Dot Blots Gene arrays consist of cDNA clones (sometimes in the form of PCR products, sometimes as oligonucleotides) or the correspond￾ing oligos spotted at high density on a nylon membrane, glass slide, or other solid support. By hybridizing labeled cDNA probes reverse transcribed from mRNA, the expression of potentially hundreds of genes can be simultaneously analyzed.This procedure requires that the labeled cDNA be present in excess of the target spotted on the array. This is difficult to achieve unless poly(A) RNA is used as template in the labeling reaction. 200 Martin et al

Ribonuclease Protection Assays Either total RNA or poly(A)RNA can be used as starting mate rial in nuclease protection assays. However, total RNa usually af- fords enough sensitivity to detect even rare messages, when the maximum amount(as much as 80 to 100 ug)is used in the assay If the gene is expressed at extremely low levels, requiring week long exposure times for detection, a switch to poly (A)RNA might prove beneficial and may justify the added cost. Although very sensitive, nuclease protection assays do require laborious gel purification of the full-length probe to avoid getting confusing RT-PCR RT-PCR is the most sensitive method for detecting and quant tating mRNA. Theoretically, even very low-abundance messages can be detected with this technique. Total RNA is routinely used as the template for RT-PCR, (Frohman, 1990) but some cloning situations and rare messages require the use of poly(A)RNA (Amersham Pharmacia Biotech, 1995) Note that one school of thought concerning RT-PCR considers it advisable to treat the sample rna with DNase I, since no purifi cation method produces RNA completely free of contaminating genomic DNA. RT-PCR is sensitive enough that even very small amounts of genomic DNA contamination can cause false posi tives. A second school of thought preaches avoidance of DNase I as discussed in Chapter 11, " PCR cDNA Library Synthesis As mentioned earlier, high-quality mRNA that is essentially free of ribosomal RNA is required for constructing CDNA libra ries. Unacceptably high backgrounds of ribosomal RNa clones would be produced if total RNa were reverse transcribed to pre pare CDNA Is It possible to predict the total rna yield from a certain Mass of tissue or number of cells? The data provided in this section are based on experimentation at Ambion, Inc. using a variety of samples and different purifica tion products. The reader is cautioned that these are theoretical estimates, and yields can vary widely based on the type of tissue or cells used for the isolation, especially when dealing with difficult samples, as discussed later. The importance of rapid and complete tissue disruption, and homogenizing at subfreezing tem RNA Purification

Ribonuclease Protection Assays Either total RNA or poly(A) RNA can be used as starting mate￾rial in nuclease protection assays. However, total RNA usually af￾fords enough sensitivity to detect even rare messages, when the maximum amount (as much as 80 to 100mg) is used in the assay. If the gene is expressed at extremely low levels, requiring week￾long exposure times for detection, a switch to poly(A) RNA might prove beneficial and may justify the added cost. Although very sensitive, nuclease protection assays do require laborious gel purification of the full-length probe to avoid getting confusing results. RT-PCR RT-PCR is the most sensitive method for detecting and quanti￾tating mRNA. Theoretically, even very low-abundance messages can be detected with this technique. Total RNA is routinely used as the template for RT-PCR, (Frohman, 1990) but some cloning situations and rare messages require the use of poly(A) RNA (Amersham Pharmacia Biotech, 1995). Note that one school of thought concerning RT-PCR considers it advisable to treat the sample RNA with DNase I, since no purifi- cation method produces RNA completely free of contaminating genomic DNA. RT-PCR is sensitive enough that even very small amounts of genomic DNA contamination can cause false posi￾tives. A second school of thought preaches avoidance of DNase I, as discussed in Chapter 11, “PCR.” cDNA Library Synthesis As mentioned earlier, high-quality mRNA that is essentially free of ribosomal RNA is required for constructing cDNA libra￾ries. Unacceptably high backgrounds of ribosomal RNA clones would be produced if total RNA were reverse transcribed to pre￾pare cDNA. Is It Possible to Predict the Total RNA Yield from a Certain Mass of Tissue or Number of Cells? The data provided in this section are based on experimentation at Ambion, Inc. using a variety of samples and different purifica￾tion products. The reader is cautioned that these are theoretical estimates, and yields can vary widely based on the type of tissue or cells used for the isolation, especially when dealing with difficult samples, as discussed later. The importance of rapid and complete tissue disruption, and homogenizing at subfreezing tem￾RNA Purification 201