关于RNA的一整套操作
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Working with RNA
Living with RNase
Most researchers are acutely aware of the risk of RNase contamination, and we do not want to belabor this point or cause undue worry. We do not routinely find it necessary to treat the microcentrifuge tubes used with RNA if they are from unopened bags or from bags in which care was taken to avoid contaminating the tubes. Yet we do consistently find a small percentage of tubes (even those marketed as being RNase-free), the use of which results in RNA degradation. We do recommend that gloves be worn when handling any reagents or reaction vessels. (Note: Gloves which have touched refrigerator handles, door knobs, or pipettors are not RNase-free.) When performing procedures that use RNases (eg. ribonuclease protection assays and plasmid purifications), care should be taken that pipettors are not contaminated by accident. One potential source of contamination is the metal tip ejector mechanism on the side of the pipettor. Removing the metal ejector bar when it is necessary to insert the pipettor into a larger vessel where the ejector could come into contact with the walls or contents of the vessel will eliminate this concern.
A. Detecting RNase
While contaminating RNase can result in a failed experiment, it is often difficult an time-consuming to determine which solution or piece of equipment is responsible. In Ambion's Quality Control Department, we use an extremely sensitive RNA probe stability assay to detect RNase contamination. This assay can be performed in your own lab to detect RNases and a protocol is provided in Technical Bulletin 166 to facilitate this. However this assay is time consuming and requires working with radioactivity. As an alternative, Ambion's RNaseAlert™ Kit (Cat #1964) allows researchers to identify contaminated reagents and equipment quickly, and nonisotopically. In the RNaseAlert Kit procedure, an optimized RNA oligonucleotide, double-labeled with both fluorescent and quenching moieties, is introduced as a target for any contaminating RNase. In the presence of RNase, the substrate is cleaved, releasing the fluor which then fluoresces. The fluorescence signal can be detected by eye or with a fluorometer.
B. Getting rid of RNase
If RNase contamination of reagents or equipment is suspected to be a problem, extra precautions may be necessary. Autoclaving tips, tubes and solutions is not sufficient to inactivate RNases. Glassware can be baked at 300°C for four hours and plasticware, tubes and most solutions can be DEPC-treated (see below). However, both procedures are time-consuming, and DEPC is both expensive and possibly carcinogenic. As an alternative, Ambion's RNaseZap™ (Catalog #9780) can be used to eliminate RNase from glassware, plastic surfaces, countertops, and pipettors. RNaseZap™ has been shown to effectively inactivate 5 µg of RNase dried onto the bottom of eppendorf tubes without inhibiting subsequent enzymatic reactions performed in the same tube. The solution contains three ingredients known to be active against RNase. RNaseZap™ can be poured onto or wiped over surfaces and works immediately upon contact. Treated labware is simply rinsed twice with distilled water and is ready for use.
How to Precipitate RNA
How to Precipitate RNA
A. Precipitating with alcohol
Precipitating RNA with alcohol (ethanol or isopropanol) requires a minimum concentration of monovalent cations (for example: 0.2 M Na+, K+; 0.5 M NH4+) (Wallace, 1987). After the salt concentration has been adjusted, the RNA may be precipitated by adding 2.5 volumes of ethanol or 1 volume of isopropanol and mixing thoroughly, followed by chilling for at least 15 minutes at -20° C. While isopropanol is somewhat less efficient at precipitating RNA, isopropanol in the presence of NH4+ is better than ethanol at keeping free nucleotides in solution, and so separating them from precipitated RNA. RNA precipitation is faster and more complete at higher RNA concentrations. A general rule of thumb is that RNA concentrations of 10 µg/ml can usually be precipitated in several hours to overnight with no difficulty, but at lower concentrations a carrier nucleic acid or glycogen should be added to facilitate precipitation and maximize recovery.
B. Precipitating with lithium chloride
Lithium Chloride may also be used to precipitate RNA, and has the advantage of not precipitating carbohydrate, protein or DNA. LiCl is frequently used to remove inhibitors of translation which copurify with RNA prepared by other methods. A final LiCl concentration of 2-3 M is needed to precipitate RNA (adding an equal volume of 4 M LiCl, 20 mM Tris-HCl, pH 7.4, and 10 mM EDTA works well). Note that no alcohol is needed for LiCl precipitation. RNA should be allowed to precipitate at -20°C; precipitation time depends on RNA concentration. It is generally safe to allow the RNA to precipitate for several hours to overnight. After centrifugation to collect the RNA, pellets can be rinsed with 70% ethanol to remove traces of LiCl. LiCl efficiently precipitates RNA greater than 300 nt in length. While LiCl can effectively precipitate RNA from more dilute solutions, for best results, the RNA concentration should exceed 200 µg/ml.
Ten Ways to Improve Your RNA Isolation
Ten Ways to Improve Your RNA Isolation
1. Immediately inactivate endogenous, intracellular RNases.
Endogenous RNases must be inactivated immediately upon tissue harvesting and cell death to prevent RNA degradation.
There are 3 effective methods to accomplish this:
i. Homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g. containing guanidinium).
ii. Flash freeze samples in liquid nitrogen. In order to inactivate RNase by flash freezing, it is important that tissue pieces be small enough to freeze almost immediately upon immersion in liquid nitrogen.
iii. Place samples in RNAlater™ Tissue Collection: RNA Stabilization Solution, an aqueous, nontoxic collection reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples (see #2 below). It is essential that tissue samples be in thin pieces (0.5 cm) so that the RNAlater can quickly permeate the tissue before RNases destroy the RNA.
2. Use proper cell or tissue storage conditions.
When samples have been flash frozen they must be stored at -80°C and never allowed to thaw. Even brief thawing prior to homogenization in a guanidinium-based lysis solution can result in RNA degradation and loss. Flash frozen tissues should be ground or pulverized at cryogenic temperatures prior to homogenization in a lysis solution.
RNAlater offers much greater flexibility for storage. Cells or tissues can be harvested into RNAlater and stored at room temperature for up to 1 week, at 4°C for up to 1 month, or at -20°C indefinitely. For more information on RNAlater, read www.ambion.com/techlib/resources/RNAlater.
3. Thoroughly homogenize samples.
Thorough homogenization of cells or tissues is an essential step in RNA isolation that prevents both RNA loss and RNA degradation. The method of homogenization should be tailored to the cell or tissue type. Whereas most cultured cells can be homogenized by simply vortexing in a cell lysis solution, animal tissues, plant tissues, yeast, and bacteria often require more rigorous methods of disruption. Bacterial cell walls for example, may require enzymatic digestion to achieve thorough cell lysis and maximum recovery of RNA. The GramCracker™ Bacterial Cell Lysis Reagents include lytic enzymes and accessory reagents to hydrolyze the N-acetyl-muramic acid linkages in the polysaccharide moiety of the cell wall. For detailed information about which disruption method is ideal for many sample types read www.ambion.com/techlib/tb/tb_183.html.
4. Pretreat homogenate before RNA isolation.
Additional treatments are needed for some samples after homogenization and before RNA isolation. Lysates made from tissues high in fat, like brain and adipose tissue, should be extracted with chloroform to remove lipids and increase RNA yields. Many plant tissues are high in polyphenolics and polysaccharides that can decrease RNA quality and yield. Pretreatment of the lysate with the Plant RNA Isolation Aid removes these troublesome compounds
5. Choose the best RNA isolation method.
With all of the different RNA isolation methods available it can be difficult to decide which one to use. The easiest and safest methods available are column-based methods like RNAqueous™ or RNAqueous-4PCR. Due to ease of handling, these procedures are ideal for working with multiple samples. When working with difficult tissues, for example ones that are high in nucleases (pancreas) or fat (brain and adipose tissue), a more rigorous, phenol-based RNA isolation method like RNAwiz™ or ToTALLY RNA™ is recommended. For additional information read www.ambion.com/techlib/tn/83/8311.html.
6. DNase treatment.
When the RNA will be used for RT-PCR, we recommend treating it with DNase to remove residual contaminating DNA. DNase treatment is also a good idea when isolating RNA from tissues that are high in DNA, like spleen. Ambion's RNAqueous-4PCR Kit includes a DNase treatment as part of the protocol, and is supplied with the necessary reagents. The DNA-free™ DNase treatment & Removal Reagents can be used to remove contaminating DNA from RNA purified by any method. Both products offer high quality DNase I, an optimized reaction buffer, and a quick and easy way to remove the DNase after the treatment without using organic solvents or risking a heat treatment.
7. Reduce exposure to environmental RNases.
To isolate intact, high quality RNA, it is essential that RNases are not introduced into RNA preparations once they are no longer protected by strong protein denaturants such as a chaotropic lysis solution or phenol. Since RNases are found almost everywhere, it is essential that any item that could contact the purified RNA is RNase-free. All surfaces, including pipettors, benchtops, glassware, and gel equipment, should be decontaminated with a surface decontamination solution like RNaseZap™ or RNaseZap Wipes. RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently.
8. Proper precipitation.
The purified RNA may need to be concentrated by precipitation for downstream applications. An ammonium acetate (NH4OAc) precipitation (0.1 volumes of 5 M NH4OAc, and 2-2.5 volumes 100% ethanol, at -20– for >25 min) gives good recovery of RNA. For quantitative recovery of low concentrations of RNA (ng/ml), an inert coprecipitant (e.g. glycogen, yeast RNA, or linear acrylamide) should be used. Linear acrylamide and DNase-treated glycogen are the coprecipitants of choice when the RNA will be used in RT-PCR because they do not contain contaminating DNA. Yeast RNA and untreated glycogen could introduce nucleic acid contamination into samples, potentially skewing RT-PCR results. After precipitation, avoid complete drying of the RNA pellet because it can make RNA difficult to resuspend.
9. Resuspension.
The final step in many RNA isolation procedures is to suspend the purified RNA pellet. The 3 ideal qualities of a resuspension solution are that it be RNase-free, have a low pH (pH 6-7), and incorporate a chelating agent to protect against RNA degradation by introduced RNases. (THE RNA Storage Solution meets all of these criteria.) To aid solubilization, the RNA pellet should be incubated in resuspension solution at 65– for 5 min with intermittent gentle vortexing.
10. Storage.
For short-term storage, resuspended RNA should be stored at -20°C; for long-term storage, it should be stored at -80°C. Although RNA resuspended in water or buffer can be stored at -80°C, RNA is most stable in an NH4OAc/ethanol precipitation mixture at -80°C. We recommend aliquotting RNA solutions into several tubes. This will both prevent damage to the RNA from successive freeze-thaw events, and help to prevent accidental RNase contamination.
How to Store RNA
How to Store RNA
RNA may be stored in a number of ways. For short-term storage, RNase-free H2O (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1mM EDTA) may be used. RNA is generally stable at -80° C for up to a year without degradation. Magnesium and other metals catalyze non-specific cleavages in RNA, and so should be chelated by the addition of EDTA if RNA is to be stored and retrieved intact. It is important to use an EDTA solution known to be RNase-free for this purpose (older EDTA solutions may have microbial growth which could contaminate the RNA sample with nucleases). It has been suggested that RNA solubilized in formamide may be stored at -20°C without degradation for at least one year (Chomczynski, 1992).
For long term storage, RNA samples may also be stored at -20°C as ethanol precipitates. Accessing these samples on a routine basis can be a nuisance, however, since the precipitates must be pelleted and dissolved in an aqueous buffer before pipetting, if accurate quantitation is important. An alternative is to pipet directly out of an ethanol precipitate that has been vortexed to create an even suspension. We have found, however, that while this method is suitable for qualitative work, it is too imprecise for use in quantitative experiments. RNA does not disperse uniformly in ethanol, probably because it forms aggregates; non-uniform suspension, in turn, leads to inconsistency in the amount of RNA removed when equal volumes are pipetted.
Increasing Your RNA Recovery During Tissue or Cell Extraction
Increasing Your RNA Recovery During Tissue or Cell Extraction
One of the most problematic steps in RNA isolation is the first step - thorough lysis of the tissue or cell sample in a denaturant solution that inhibits RNA degradation by RNase. While it is possible to process fresh tissue directly, it is extremely important that all cells are disrupted immediately upon contact with the denaturant. This usually requires use of a polytron and even then some "difficult to process tissues" (e.g. hard tumors, bacterial cells, plant roots, etc.) are not effectively disrupted (see the article, "Cell Disruption: Getting the RNA Out "). Therefore, if you are having a problem with yield or degradation during RNA isolation, we usually recommend freezing the tissue sample before processing. Here we compare three methods for processing frozen tissues in a side-by-side test for quantity of mRNA recovered.
Freezing the Tissue
Samples should be frozen quickly so that the whole tissue sample freezes at once throughout. This may mean mincing the tissue into smaller fragments before freezing. Submerging the samples in liquid nitrogen will freeze the tissue pieces most quickly. Alternatively, a metal plate placed on dry ice can serve as a freezing surface.
Each of the methods below describes a distinct way of generating a tissue/cell lysate from which to purify RNA and is assessed for yield of poly(A+)RNA, when used to process 0.1 g of frozen mouse liver tissue. While the three methods each use a guanidine buffer to ultimately lyse the cells, they differ in how the tissue is processed prior or during that lysis step.
Method 1: Processing frozen tissue fragments in a dounce
Yield: 4.1 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, placed in a dounce, and processed as lysis buffer is added. Both pestle A and pestle B are used for ten strokes each.
Method 2: Processing frozen tissue fragments through a syringe
Yield: 3.2 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, added to lysis buffer and passed back and forth ten times through an 18 gauge syringe needle.
Method 3: Grinding the tissue to a powder in liquid N2
Yield: 7.1 µg poly(A+)RNA
The frozen sample is powdered by grinding the frozen tissue fragments in a prechilled mortar and occasionally adding liquid N2 into the mortar to prevent thawing. Once the tissue is ground to a fine powder, the denaturing solution is added to the mortar, and the semi-frozen mixture is stirred. This mixture can then be thawed and transferred to an appropriate vessel for further processing.
Note that by grinding the tissue to a powder in liquid N2 (Method 3), cellular disruption is much more complete resulting almost twice the yield of the other two methods.
Reprinted from Ambion's TechNotes Newsletter 3:3, © 1998
Treating Solutions with DEPC to Remove RNase
Treating Solutions with DEPC to Remove RNase
To ensure that solutions are free of RNase contamination, they can be treated with diethylpyrocarbonate (DEPC) [WARNING: DEPC is a suspected carcinogen: Take appropriate precautions when handling; e.g., always wear gloves and handle under an approved fume hood]. DEPC reacts with histidine residues of proteins and will inactivate RNases. However, it can also react with RNA, so it needs to be removed by heat treatment before the solution is used (DEPC breaks down to CO2 and ethanol). Add DEPC to solutions at a concentration of 0.05 - 0.1% (e.g., add 0.5 - 1 ml DEPC per liter); stir or shake into solution, incubate for several hours; autoclave at least 45 minutes, or until DEPC scent is gone. Please be aware that compounds containing primary amine groups, such as Tris (2-Amino-2-hydroxymethyl-1,3-propanediol), will also react with DEPC, and thus should be added only after DEPC treatment is complete. Note: We have observed that distilled water, treated with DEPC and thoroughly autoclaved, caused a 20% inhibition of translation in a reticulocyte lysate. We find that distilled water is generally already RNase-free, and so does not need to be treated.
Living with RNase
Most researchers are acutely aware of the risk of RNase contamination, and we do not want to belabor this point or cause undue worry. We do not routinely find it necessary to treat the microcentrifuge tubes used with RNA if they are from unopened bags or from bags in which care was taken to avoid contaminating the tubes. Yet we do consistently find a small percentage of tubes (even those marketed as being RNase-free), the use of which results in RNA degradation. We do recommend that gloves be worn when handling any reagents or reaction vessels. (Note: Gloves which have touched refrigerator handles, door knobs, or pipettors are not RNase-free.) When performing procedures that use RNases (eg. ribonuclease protection assays and plasmid purifications), care should be taken that pipettors are not contaminated by accident. One potential source of contamination is the metal tip ejector mechanism on the side of the pipettor. Removing the metal ejector bar when it is necessary to insert the pipettor into a larger vessel where the ejector could come into contact with the walls or contents of the vessel will eliminate this concern.
A. Detecting RNase
While contaminating RNase can result in a failed experiment, it is often difficult an time-consuming to determine which solution or piece of equipment is responsible. In Ambion's Quality Control Department, we use an extremely sensitive RNA probe stability assay to detect RNase contamination. This assay can be performed in your own lab to detect RNases and a protocol is provided in Technical Bulletin 166 to facilitate this. However this assay is time consuming and requires working with radioactivity. As an alternative, Ambion's RNaseAlert™ Kit (Cat #1964) allows researchers to identify contaminated reagents and equipment quickly, and nonisotopically. In the RNaseAlert Kit procedure, an optimized RNA oligonucleotide, double-labeled with both fluorescent and quenching moieties, is introduced as a target for any contaminating RNase. In the presence of RNase, the substrate is cleaved, releasing the fluor which then fluoresces. The fluorescence signal can be detected by eye or with a fluorometer.
B. Getting rid of RNase
If RNase contamination of reagents or equipment is suspected to be a problem, extra precautions may be necessary. Autoclaving tips, tubes and solutions is not sufficient to inactivate RNases. Glassware can be baked at 300°C for four hours and plasticware, tubes and most solutions can be DEPC-treated (see below). However, both procedures are time-consuming, and DEPC is both expensive and possibly carcinogenic. As an alternative, Ambion's RNaseZap™ (Catalog #9780) can be used to eliminate RNase from glassware, plastic surfaces, countertops, and pipettors. RNaseZap™ has been shown to effectively inactivate 5 µg of RNase dried onto the bottom of eppendorf tubes without inhibiting subsequent enzymatic reactions performed in the same tube. The solution contains three ingredients known to be active against RNase. RNaseZap™ can be poured onto or wiped over surfaces and works immediately upon contact. Treated labware is simply rinsed twice with distilled water and is ready for use.
How to Precipitate RNA
How to Precipitate RNA
A. Precipitating with alcohol
Precipitating RNA with alcohol (ethanol or isopropanol) requires a minimum concentration of monovalent cations (for example: 0.2 M Na+, K+; 0.5 M NH4+) (Wallace, 1987). After the salt concentration has been adjusted, the RNA may be precipitated by adding 2.5 volumes of ethanol or 1 volume of isopropanol and mixing thoroughly, followed by chilling for at least 15 minutes at -20° C. While isopropanol is somewhat less efficient at precipitating RNA, isopropanol in the presence of NH4+ is better than ethanol at keeping free nucleotides in solution, and so separating them from precipitated RNA. RNA precipitation is faster and more complete at higher RNA concentrations. A general rule of thumb is that RNA concentrations of 10 µg/ml can usually be precipitated in several hours to overnight with no difficulty, but at lower concentrations a carrier nucleic acid or glycogen should be added to facilitate precipitation and maximize recovery.
B. Precipitating with lithium chloride
Lithium Chloride may also be used to precipitate RNA, and has the advantage of not precipitating carbohydrate, protein or DNA. LiCl is frequently used to remove inhibitors of translation which copurify with RNA prepared by other methods. A final LiCl concentration of 2-3 M is needed to precipitate RNA (adding an equal volume of 4 M LiCl, 20 mM Tris-HCl, pH 7.4, and 10 mM EDTA works well). Note that no alcohol is needed for LiCl precipitation. RNA should be allowed to precipitate at -20°C; precipitation time depends on RNA concentration. It is generally safe to allow the RNA to precipitate for several hours to overnight. After centrifugation to collect the RNA, pellets can be rinsed with 70% ethanol to remove traces of LiCl. LiCl efficiently precipitates RNA greater than 300 nt in length. While LiCl can effectively precipitate RNA from more dilute solutions, for best results, the RNA concentration should exceed 200 µg/ml.
Ten Ways to Improve Your RNA Isolation
Ten Ways to Improve Your RNA Isolation
1. Immediately inactivate endogenous, intracellular RNases.
Endogenous RNases must be inactivated immediately upon tissue harvesting and cell death to prevent RNA degradation.
There are 3 effective methods to accomplish this:
i. Homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g. containing guanidinium).
ii. Flash freeze samples in liquid nitrogen. In order to inactivate RNase by flash freezing, it is important that tissue pieces be small enough to freeze almost immediately upon immersion in liquid nitrogen.
iii. Place samples in RNAlater™ Tissue Collection: RNA Stabilization Solution, an aqueous, nontoxic collection reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples (see #2 below). It is essential that tissue samples be in thin pieces (0.5 cm) so that the RNAlater can quickly permeate the tissue before RNases destroy the RNA.
2. Use proper cell or tissue storage conditions.
When samples have been flash frozen they must be stored at -80°C and never allowed to thaw. Even brief thawing prior to homogenization in a guanidinium-based lysis solution can result in RNA degradation and loss. Flash frozen tissues should be ground or pulverized at cryogenic temperatures prior to homogenization in a lysis solution.
RNAlater offers much greater flexibility for storage. Cells or tissues can be harvested into RNAlater and stored at room temperature for up to 1 week, at 4°C for up to 1 month, or at -20°C indefinitely. For more information on RNAlater, read www.ambion.com/techlib/resources/RNAlater.
3. Thoroughly homogenize samples.
Thorough homogenization of cells or tissues is an essential step in RNA isolation that prevents both RNA loss and RNA degradation. The method of homogenization should be tailored to the cell or tissue type. Whereas most cultured cells can be homogenized by simply vortexing in a cell lysis solution, animal tissues, plant tissues, yeast, and bacteria often require more rigorous methods of disruption. Bacterial cell walls for example, may require enzymatic digestion to achieve thorough cell lysis and maximum recovery of RNA. The GramCracker™ Bacterial Cell Lysis Reagents include lytic enzymes and accessory reagents to hydrolyze the N-acetyl-muramic acid linkages in the polysaccharide moiety of the cell wall. For detailed information about which disruption method is ideal for many sample types read www.ambion.com/techlib/tb/tb_183.html.
4. Pretreat homogenate before RNA isolation.
Additional treatments are needed for some samples after homogenization and before RNA isolation. Lysates made from tissues high in fat, like brain and adipose tissue, should be extracted with chloroform to remove lipids and increase RNA yields. Many plant tissues are high in polyphenolics and polysaccharides that can decrease RNA quality and yield. Pretreatment of the lysate with the Plant RNA Isolation Aid removes these troublesome compounds
5. Choose the best RNA isolation method.
With all of the different RNA isolation methods available it can be difficult to decide which one to use. The easiest and safest methods available are column-based methods like RNAqueous™ or RNAqueous-4PCR. Due to ease of handling, these procedures are ideal for working with multiple samples. When working with difficult tissues, for example ones that are high in nucleases (pancreas) or fat (brain and adipose tissue), a more rigorous, phenol-based RNA isolation method like RNAwiz™ or ToTALLY RNA™ is recommended. For additional information read www.ambion.com/techlib/tn/83/8311.html.
6. DNase treatment.
When the RNA will be used for RT-PCR, we recommend treating it with DNase to remove residual contaminating DNA. DNase treatment is also a good idea when isolating RNA from tissues that are high in DNA, like spleen. Ambion's RNAqueous-4PCR Kit includes a DNase treatment as part of the protocol, and is supplied with the necessary reagents. The DNA-free™ DNase treatment & Removal Reagents can be used to remove contaminating DNA from RNA purified by any method. Both products offer high quality DNase I, an optimized reaction buffer, and a quick and easy way to remove the DNase after the treatment without using organic solvents or risking a heat treatment.
7. Reduce exposure to environmental RNases.
To isolate intact, high quality RNA, it is essential that RNases are not introduced into RNA preparations once they are no longer protected by strong protein denaturants such as a chaotropic lysis solution or phenol. Since RNases are found almost everywhere, it is essential that any item that could contact the purified RNA is RNase-free. All surfaces, including pipettors, benchtops, glassware, and gel equipment, should be decontaminated with a surface decontamination solution like RNaseZap™ or RNaseZap Wipes. RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently.
8. Proper precipitation.
The purified RNA may need to be concentrated by precipitation for downstream applications. An ammonium acetate (NH4OAc) precipitation (0.1 volumes of 5 M NH4OAc, and 2-2.5 volumes 100% ethanol, at -20– for >25 min) gives good recovery of RNA. For quantitative recovery of low concentrations of RNA (ng/ml), an inert coprecipitant (e.g. glycogen, yeast RNA, or linear acrylamide) should be used. Linear acrylamide and DNase-treated glycogen are the coprecipitants of choice when the RNA will be used in RT-PCR because they do not contain contaminating DNA. Yeast RNA and untreated glycogen could introduce nucleic acid contamination into samples, potentially skewing RT-PCR results. After precipitation, avoid complete drying of the RNA pellet because it can make RNA difficult to resuspend.
9. Resuspension.
The final step in many RNA isolation procedures is to suspend the purified RNA pellet. The 3 ideal qualities of a resuspension solution are that it be RNase-free, have a low pH (pH 6-7), and incorporate a chelating agent to protect against RNA degradation by introduced RNases. (THE RNA Storage Solution meets all of these criteria.) To aid solubilization, the RNA pellet should be incubated in resuspension solution at 65– for 5 min with intermittent gentle vortexing.
10. Storage.
For short-term storage, resuspended RNA should be stored at -20°C; for long-term storage, it should be stored at -80°C. Although RNA resuspended in water or buffer can be stored at -80°C, RNA is most stable in an NH4OAc/ethanol precipitation mixture at -80°C. We recommend aliquotting RNA solutions into several tubes. This will both prevent damage to the RNA from successive freeze-thaw events, and help to prevent accidental RNase contamination.
How to Store RNA
How to Store RNA
RNA may be stored in a number of ways. For short-term storage, RNase-free H2O (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1mM EDTA) may be used. RNA is generally stable at -80° C for up to a year without degradation. Magnesium and other metals catalyze non-specific cleavages in RNA, and so should be chelated by the addition of EDTA if RNA is to be stored and retrieved intact. It is important to use an EDTA solution known to be RNase-free for this purpose (older EDTA solutions may have microbial growth which could contaminate the RNA sample with nucleases). It has been suggested that RNA solubilized in formamide may be stored at -20°C without degradation for at least one year (Chomczynski, 1992).
For long term storage, RNA samples may also be stored at -20°C as ethanol precipitates. Accessing these samples on a routine basis can be a nuisance, however, since the precipitates must be pelleted and dissolved in an aqueous buffer before pipetting, if accurate quantitation is important. An alternative is to pipet directly out of an ethanol precipitate that has been vortexed to create an even suspension. We have found, however, that while this method is suitable for qualitative work, it is too imprecise for use in quantitative experiments. RNA does not disperse uniformly in ethanol, probably because it forms aggregates; non-uniform suspension, in turn, leads to inconsistency in the amount of RNA removed when equal volumes are pipetted.
Increasing Your RNA Recovery During Tissue or Cell Extraction
Increasing Your RNA Recovery During Tissue or Cell Extraction
One of the most problematic steps in RNA isolation is the first step - thorough lysis of the tissue or cell sample in a denaturant solution that inhibits RNA degradation by RNase. While it is possible to process fresh tissue directly, it is extremely important that all cells are disrupted immediately upon contact with the denaturant. This usually requires use of a polytron and even then some "difficult to process tissues" (e.g. hard tumors, bacterial cells, plant roots, etc.) are not effectively disrupted (see the article, "Cell Disruption: Getting the RNA Out "). Therefore, if you are having a problem with yield or degradation during RNA isolation, we usually recommend freezing the tissue sample before processing. Here we compare three methods for processing frozen tissues in a side-by-side test for quantity of mRNA recovered.
Freezing the Tissue
Samples should be frozen quickly so that the whole tissue sample freezes at once throughout. This may mean mincing the tissue into smaller fragments before freezing. Submerging the samples in liquid nitrogen will freeze the tissue pieces most quickly. Alternatively, a metal plate placed on dry ice can serve as a freezing surface.
Each of the methods below describes a distinct way of generating a tissue/cell lysate from which to purify RNA and is assessed for yield of poly(A+)RNA, when used to process 0.1 g of frozen mouse liver tissue. While the three methods each use a guanidine buffer to ultimately lyse the cells, they differ in how the tissue is processed prior or during that lysis step.
Method 1: Processing frozen tissue fragments in a dounce
Yield: 4.1 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, placed in a dounce, and processed as lysis buffer is added. Both pestle A and pestle B are used for ten strokes each.
Method 2: Processing frozen tissue fragments through a syringe
Yield: 3.2 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, added to lysis buffer and passed back and forth ten times through an 18 gauge syringe needle.
Method 3: Grinding the tissue to a powder in liquid N2
Yield: 7.1 µg poly(A+)RNA
The frozen sample is powdered by grinding the frozen tissue fragments in a prechilled mortar and occasionally adding liquid N2 into the mortar to prevent thawing. Once the tissue is ground to a fine powder, the denaturing solution is added to the mortar, and the semi-frozen mixture is stirred. This mixture can then be thawed and transferred to an appropriate vessel for further processing.
Note that by grinding the tissue to a powder in liquid N2 (Method 3), cellular disruption is much more complete resulting almost twice the yield of the other two methods.
Reprinted from Ambion's TechNotes Newsletter 3:3, © 1998
Treating Solutions with DEPC to Remove RNase
Treating Solutions with DEPC to Remove RNase
To ensure that solutions are free of RNase contamination, they can be treated with diethylpyrocarbonate (DEPC) [WARNING: DEPC is a suspected carcinogen: Take appropriate precautions when handling; e.g., always wear gloves and handle under an approved fume hood]. DEPC reacts with histidine residues of proteins and will inactivate RNases. However, it can also react with RNA, so it needs to be removed by heat treatment before the solution is used (DEPC breaks down to CO2 and ethanol). Add DEPC to solutions at a concentration of 0.05 - 0.1% (e.g., add 0.5 - 1 ml DEPC per liter); stir or shake into solution, incubate for several hours; autoclave at least 45 minutes, or until DEPC scent is gone. Please be aware that compounds containing primary amine groups, such as Tris (2-Amino-2-hydroxymethyl-1,3-propanediol), will also react with DEPC, and thus should be added only after DEPC treatment is complete. Note: We have observed that distilled water, treated with DEPC and thoroughly autoclaved, caused a 20% inhibition of translation in a reticulocyte lysate. We find that distilled water is generally already RNase-free, and so does not need to be treated.
