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I precipitated bacterial RNA during 1 hour at -80ºC after depleting rRNA with Ribo-Zero kit.
Does more time lead to better results?
As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a very close estimate.
Interestingly neither the time nor the temperature of a precipitation have a great influence on the outcome - they change the yield only by a few percent. If you look on figure 2 from the paper mentioned, you see the following:
This subfigure shows that the recovery is almost not influenced by the temperature, but only by the conconcentration of the DNA (or RNA). Depending on how much RNA you expect (how much cells did you use for the isolation) I would add a co-precipitation agent when you have only very small amounts on RNA. Glycogen works very well here. Otherwise I wouldn't worry.
This figure shows that the incubation time has only an influence on the percentage of the recovery when then time is too short. If you incubate for an hour, you are on the safe side.
As many kits suggest, RNA concentration has the most profound influence on precipitation efficiency / recovery fraction. Time, temperature and precipitation agent concentration has lesser effect. See for example instruction by Life Technologies on LiCl precipitation:
The Use of LiCl Precipitation for RNA Purification
MRNA Purification (E2065)
The kit includes LiCl solution for quick recovery of the synthesized mRNA. LiCl precipitation of RNA is effective in removing the majority of unincorporated NTPs and enzymes. However, RNAs shorter than 300 bases or at concentrations lower than 0.1 mg/ml do not precipitate well. In such cases, other purification methods may be used. LiCl purified mRNA is suitable for transfection and microinjection experiments.
- To the 20 µl transcription reaction, add 30 &mul water and 25 &mul LiCl solution, mix well.
- Incubate at &ndash20°C for 30 minutes.
- Centrifuge at 4°C for 15 minutes at top speed to pellet the RNA.
- Remove the supernatant carefully.
- Rinse the pellet by adding 500 &mul of cold 70% ethanol and centrifuge at 4°C for 10 minutes.
- Remove the ethanol carefully. Spin the tube briefly to bring down any liquid on the wall.
- Remove residual liquid carefully using a sharp tip (e.g., loading tip).
- Air dry the pellet and resuspend the mRNA in 50 &mul of 0.1 mM EDTA or a suitable RNA storage solution.
- Heat the RNA at 65°C for 5-10 minutes to completely dissolve the RNA. Mix well.
- Store the RNA at &ndash20°C or below.
Phenol-chloroform Extraction and Ethanol Precipitation
For removal of proteins and most of the free nucleotides, phenol:chloroform extraction and ethanol precipitation of RNA transcripts is the preferred method.
- Adjust the reaction volume to 180 &mul by adding nuclease-free water. Add 20 &mul of 3 M sodium acetate, pH 5.2 or 20 &mul of 5 M ammonium acetate and mix thoroughly.
- Extract with an equal volume of 1:1 phenol:chloroform mixture, followed by two extractions with chloroform. Collect the aqueous phase and transfer to a new tube.
- Precipitate the RNA by adding 2 volumes of ethanol. Incubate at &ndash20°C for at least 30 minutes and collect the pellet by centrifugation.
- Remove the supernatant and rinse the pellet with 500 &mul of ice cold 70% ethanol.
- Resuspend the RNA in 50 &mul 0.1 mM EDTA. Store the RNA at &ndash20°C or below.
Double aspiration is useful for removing the last traces of EtOH supernatant after precipitations. It involves a second quick spin and aspiration to ensure removal of any precipitation supernatant e.g. on the walls of the tube, that might interfere with downstream steps of the protocol. We recommend it in Ambion's RPA III™ protocol, and for RNA probe template preparation.
- After pelleting the precipitation, aspirate the precipitation supernatant off the nucleic acid pellet. Follow immediately with a quick 1–2 second centrifugation and aspirate again.
Aspiration can be done with a syringe needle or a drawn-out Pasteur pipette connected to a vacuum source with a trap. Alternatively, a drawn-out Pasteur pipette can be used with a pipette bulb. To make drawn-out Pasteur pipettes, soften the pipette tip with a flame and draw the tip out with forceps, break the tip at the narrowest point and flame polish if needed.
The source of contamination by RNases during RNA extraction can be exogenous or endogenous 5-7 . Exogenous sources include the reagents, glassware, and plasticware used in RNA isolation, especially the skin of the investigator. However, these RNases can be eliminated through sensible measures, such as treatment of reagents and plastic utensils with diethyl pyrocarbonate (DEPC) baking the glassware, mortar and pestle and wearing disposable gloves throughout the whole procedure. However, endogenous RNases are innate to biological tissues and are normally sequestered in organelles and vacuoles. They are highly regulated in intact cells, and the regulatory mechanisms are destroyed once the organelles and vacuoles are disrupted during cell lysis, which could lead to rapid degradation of RNA 5 . Therefore, how to quickly and completely inactivate the released endogenous RNases is the key to successful purification of high-quality RNA. Guanidinium salts have been shown to be powerful inhibitors of RNases 5, 7 and many guanidinium salt-based methods have been established for RNA isolation. However, some drawbacks still exist for these methods, such as RNA loss or fragmentation during organic extraction, interference of downstream enzymatic reactions by guanidinium salt residue, and a higher cost for the experiment because of the using of a higher concentration of guanidinium salts to inactivate RNases 7 . In our previously reported RNA isolation method 6 , we tried to quickly and completely inactivate endogenous RNases released during cell lysis as well as overcome some drawbacks of guanidinium salt-based methods. Our measures included modifying the composition of the extraction buffer: the pH of the extraction buffer was not adjusted with HCl, and the final pH, including 0.2 m Tris, in our system is 9.0. This pH could greatly reduce RNase activity because only less than 15% of the activity remains when the pH is above 9.0 compared with the maximum activity at a pH of approximately 7.2 in water 10 . In addition, MgCl2 and sucrose were included in the extraction buffer. The MgCl2 was added because Mg 2+ are needed to stabilize many secondary and tertiary structures within the RNA 11 , and the sucrose was added to maintain an appropriate osmotic pressure for the solution. Otherwise in hypotonic solution, the fragmentation of RNA might occur through impingement of the osmotic pressure on RNAs on the explosive cell lysis. Furthermore, Tris-saturated phenol was added immediately into liquid nitrogen-ground tissue powders to inactivate the released endogenous RNases. During the following organic extraction, vortexing was adopted to enhance the effects of protein denaturation. Inclusion of MgCl2 and sucrose in extraction buffer could also avoid the potential threat of fragmentation of RNAs, especially those with high molecular weight by vortexing. To reduce RNase activity introduced accidently during the whole isolation procedure, all reagents used for RNA preparation were ice-cold, the tubes were prechilled, and the samples were maintained on ice at all times, except during steps for air drying the nucleic acid sediments after centrifugation 5 .
Nucleic acid concentration is usually the final step in most RNA purification protocols. Normally, the RNA concentration is achieved by precipitation in the presence of sodium ions and ethanol. However, unlike the dramatic precipitation of genomic DNA, longer incubation periods at −20 °C are often required for RNA precipitation to ensure complete recovery 5 . In our original protocol, nucleic acid recovery was achieved through ethanol precipitation at −20 °C for 3 hr, and included two ethanol precipitation steps, with one to precipitate nucleic acids, including both RNA and DNA, and the other to selectively precipitate RNA 6 . The whole isolation procedure lasted for approximately 8 hr and was divided into three sessions by two longer ethanol precipitations, which makes the method unsuitable for time-limited undergraduate experimental course. There is evidence showing that the recovery of nucleic acids by ethanol precipitation is not significantly enhanced by long or low temperature incubation, but by longer centrifugation time 9 . Therefore, the longer low temperature incubation (at −20 °C for 3 hr) was replaced by incubation at 0 °C for 10 min, and the centrifugation time following ethanol precipitation were all set to 15 min to achieve good recovery. As a result, the duration of the isolation procedure was substantially reduced (in 2.5 hr, compared to 8 hr of our original method), while the recovery of RNA was comparable to our original method (data not shown).
Because the success of many downstream RNA-based applications is relied on obtaining high-quality RNA, it is necessary to assess the quality of purified RNA before conducting downstream assays. Therefore, the quantity, purity, and integrity of RNA samples should be checked with appropriate methods. RNA has a maximum absorption at 260 nm, and the RNA concentration could be quantified spectrophotometrically by the relationship 1 A260 = 40 µg RNA mL −1 . Contaminated proteins and polysaccharides/polyphenols have maximum absorption values at 280 and 230 nm, respectively, so the ratios of A260/A280 and A260/A230 could be used as indications of these contaminants. Additionally, these contaminants could result in deviations from the standard absorbance spectrum with the characteristic UV absorption profile of pure RNA samples, which is not observable when only measuring absorbance at the three wavelengths above. Therefore, besides absorption ratios, the UV absorbance spectrum analysis of purified RNA from 220 to 350 nm was introduced to assess the purity of RNA. To check RNA integrity, the typical method is to visualize the banding profile of size-separated RNA bands through an agarose gel under denaturing conditions. However, it is a time-consuming process because of the complicated procedure, and the visualization of RNA bands cannot be achieved immediately after electrophoresis either 8 . A standard agarose gel, although it has lower resolution for nondenatured RNAs rich in secondary and tertiary structures, has the advantages of being easy to complete and capable of visualizing RNA immediately after electrophoresis by including ethidium bromide (EtBr) in the gel. Therefore, electrophoresis of nondenatured RNA through a standard agarose gel was used to check RNA integrity in this report. However, it should be noted that the best performance of the separation of total RNA under nondenaturing conditions could only be achieved by running RNA samples with lower amounts (usually less than 5 μg) through a higher concentration of agarose gel (usually 1.5%).
Students worked in pairs in the lab. The RNase-free treatment was performed before the experiment by individual students. Approximately 1 hr of prelab lecture and discussion focused on specific precautions for working with RNA, and the rationale for establishing the original protocol and the modifications made for the methods of RNA isolation and RNA quality control analysis used in this report were provided and followed by a 4 hr hands-on lab exercise. The whole procedure could also be divided into two sessions of RNA isolation and RNA quality control analysis.
Materials and Solutions
The following equipment is required for the experiment: mortar and pestle, stainless lab spatulas, 50-mL centrifuge tubes, pipettes, vortex, disposable tips and gloves, centrifuge (Thermo Fisher Scientific, Am Kalkberg, Germany, Sorvall ST 16R), 1.5 mL Eppendorf tubes, low-volume spectrophotometer (NanoDrop Technologies, Wilmington, DE, Nanodrop 2000c), submarine electrophoresis facilities (Bio-Rad, Hercules, CA, Sub Cell GT), and gel imaging system (Bio-Rad, Hercules, CA, Gel Doc XR system). The RNase-free treatment was performed as follows: all tubes and tips were soaked in 0.1% DEPC overnight at 37 °C, then autoclaved at 121 °C for 20 min. The mortars and pestles as well as the stainless lab spatulas were baked at 180 °C for 12 hr.
The following reagents were required for RNA isolation: RNA extraction buffer (0.2 m Tris, 0.4 m KCl, 0.2 m sucrose, 35 m m MgCl2, 25 m m EGTA), Tris-saturated phenol (pH 8.0), chloroform/isoamyl alcohol (24:1, v/v), 3 m NaAc (pH 5.2), 3 m NaAc (pH 5.6), 0.3 m NaAc (pH 5.6), and DEPC-treated water. NaAc solutions and DEPC-treated water were prepared by treating NaAc solutions and water with 0.1% DEPC overnight at 37 °C, and then autoclaved at 121 °C for 20 min. The RNA extraction buffer was prepared with DEPC-treated water.
The following reagents were required for RNA analysis: agarose, 1 × TAE, 10 × RNA loading buffer (1 m m EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol), and 10 mg/mL EtBr. Solutions for RNA electrophoresis were not administered the RNase-free treatment because RNase and RNA bands have different mobility in agarose gels, which means they could be separated once an electric field is applied.
Cell Lysis, Nucleoprotein Dissociation, Protein Denaturation, and Removal
The following procedure can be applied to a wide variety of herbaceous plant tissues. For plant tissues rich in polysaccharides and/or polyphenols, specific purification protocols should be adopted, such as the modified cetyltrimethylammonium bromide method 12 .
Collect 1 g of Chinese white cabbage (Brassica campestris L. ssp. chinensis Makino [var. communis Tsen et Lee]) leaves and grind them in liquid nitrogen in a precooled mortar to a fine powder. Do not let the tissue thaw and immediately transfer the powder to a 50-mL centrifuge tube, add 2 mL of Tris-saturated phenol (pH 8.0) immediately, then 4 mL of extraction buffer and 2 mL of chloroform/isoamyl alcohol (24:1, v/v) sequentially (phenol should be added first to establish a denaturation environment for the endogenous RNases to be released into). Vortex the tube until a complete emulsion was formed. Centrifuge at 8,000 × g for 5 min at 4 °C. Carefully transfer aqueous phase to another 50-mL centrifuge tube, then add 2 mL of phenol and 2 mL of chloroform/isoamyl alcohol (24:1, v/v), mix vigorously and centrifuge at 8,000 × g for 5 min at 4 °C. Take the upper phase and add 4 mL of chloroform/isoamyl alcohol (24:1, v/v), then mix and centrifuge the sample as before. Transfer the upper phase to a 50-mL centrifuge tube and record the volume that was transferred.
Selective RNA Precipitation
Combinations of ethanol and NaAc (pH 5.6) were used to selectively precipitate the RNA. First, add 0.1 volume of NaAc (pH 5.2) and 2.2 volumes of ethanol precooled at −20 °C to the transferred aqueous phase and mix it by inversion several times. After embedding the tubes in crushed ice for 10 min, centrifuge them at 15,000 × g for 15 min at 4 °C. The nucleic acids were spun onto the wall of the centrifuge tube. After centrifugation, decant the liquid and mark the location of nucleic acid sediments on the outer wall of the tube. Invert the tube and place it on a filer paper to allow it to air-dry to remove residual ethanol. Resuspend the nucleic acid sediments with 1 mL of 3 m NaAc (pH 5.6) using a pipette, then transfer the resuspended nucleic acid to a 1.5 mL Eppendorf tube. Centrifuge the tube at 15,000 × g for 10 min at 4 °C, carefully decant and discard the supernatant, place the tube invertedly on a filter paper to air dry the nucleic acids. Redissolve the sediment in 400 μL of 0.3 m NaAc (pH 5.6) and add 1 mL of ethanol precooled at −20 °C, mix the tube by inversion several times, embed the tube in crushed ice for 10 min, then centrifuge the tube at 15,000 × g for 15 min at 4 °C. Wash the RNA pellet twice with 200 μL of 70% ethanol, then air-dry and dissolve the pellet in 50 μL of DEPC-treated water.
RNA Quality Assessment by Spectrophotometer
The RNA was analyzed on a NanoDrop 2000c spectrophotometer according to the manufacturer's instructions. An absorbance spectrum was obtained from 220 to 350 nm, the RNA concentration was calculated with the equation 1 A260 = 40 µg RNA mL −1 , and ratios of A260/A280 and A260/A230 were calculated to evaluate the purity of the RNA samples that were extracted.
Agarose Gel Electrophoresis
Dilute 10 μL of RNA samples to 1 µg/μL with DEPC-treated water according to the quantification results, 0.5, 1, 2, and 4 µg of RNA aliquots were taken out and adjusted to 9 µL with DEPC-treated water, then 1 µL of 10 × RNA loading buffer was added. After mixing the samples, all aliquots were loaded onto a 1.5% TAE agarose gel containing 0.5 µg/mL EtBr. Electrophoresis was carried out in 1 × TAE at 5 V/cm until the dye front had migrated two-thirds of the way down the gel. The gel was photographed with a Gel Doc XR System (Bio-Rad).
Assessment of Student Learning
Three methods were applied to assess student learning from the experimental exercises: Lab reports after the experiment, a lab presentation at the beginning of the next experimental class, and a short summary after the whole experimental course. In their lab reports, students were required to state the purpose and the principles of the experiment, concisely state the results that were obtained, including the yield in μg RNA/g of fresh weight of the leaves, the ratios of OD260/OD280 and OD260/OD230, and the results of the electrophoresis of nondenatured RNAs in a standard agarose gel in a figure, as well as state the key of the protocol design and the experimental operation, especially how to prevent RNase contamination, and state what conclusions they can make based on their data. In addition, six groups out of 15 were randomly selected to give a presentation before the whole class about their results and experiment. Thus, all of the groups could mutually compare their results and, if necessary, discuss their experimental experience. In their summary, they should list what they had learned, including their grasp of experimental skills and reinforcement of the fundamental concepts.
The liquid nitrogen used for freezing plant tissues needs to be handled carefully. Phenol and chloroform can easily evaporate, even at room temperature, and these two reagents as well as their vapors are corrosive to the eyes, the skin, and the respiratory tract. EtBr is mutagenic and should be used with caution. Phenol, chloroform, and DEPC are carcinogenic and should be handled with extreme care. Students were required to wear lab coats and closed-toed shoes in the lab as well as disposable gloves throughout the whole procedure.
The procedure is suitable for all types of tissues from wide variety of animal (and blood) and plant species. All steps are performed at room temperature (RT) (without ice) and without DEPC-treated water. RNA precipitate with lithium chloride (LiCl) for increased stability of the RNA preparation and improvement of cDNA synthesis. The following protocol is designed for small and large tissue samples (tissue volume 10-200 μl), which normally yield about 10-500 &mug of total RNA.
Materials for total RNA isolation
- (40%w/w Phenol (saturated at pH 4.3), 1 M guanidine thiocyanate, 1 M Ammonium thiocyanate, 0.1 M sodium acetate buffer (pH 5.0), 5%w/w glycerol)
- Chloroform-isoamyl alcohol mix (24:1)
- 100% isopropanol (isopropyl alcohol, 2-propanol)
- 70% ethanol
- 10 M LiCl
- Fresh Milli-Q water (or Milli-Q ultrapure BioPak water) or autoclaved 1xTE (0.1 mM EDTA, 10 mM Tris-HCl, pH 7.0). When an ultrafiltration cartridge (BioPak) is utilized at the point-of-use, the water is suitable for genomics applications (quality at least equivalent to DEPC-treated water) and cell culture.
- 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz.
- In 2 ml tube with mechanically disrupted tissue sample add fresh 1 ml TRIzol Reagent, vortex very well, and incubate the sample for 5 minutes at room temperature.
- Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent used for homogenization. Vortex very well, and incubate the sample for 3 minutes at room temperature.
- Centrifugate the samples at maximum speed on table microcentrifuge for 5 minutes at +4°C.
- Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with an equal volume of chloroform, vortex well. Spin at maximum speed on table microcentrifuge for 5 minutes.
- Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with an equal volume of 2-propanol, vortex well. Spin at maximum speed on table microcentrifuge at room temperature for 10 minutes at +4°C. Wash the pellet once with 1.5 ml 70% ethanol. Spin at maximum speed on table microcentrifuge for 5 minutes.
- Dissolve the pellet (do not dry) in 400 μl 1xTE at 55°С about 10 min, with vortex. Add an equal volume of 10 M LiCl and chill the solution at -20°C for several hours (overnight). Spin at maximum speed on table microcentrifuge for 10 minutes at +4°C. Carefully remove and discard (or save, Fig.1) supernatant (contains: small RNA < 200 nt and DNA). Wash pellet with 1.5 ml 70% ethanol, vortex well, microcentrifuge, discard the ethanol, don't dry the pellet. Dissolve the pellet in 200-400 μl fresh milliQ water (BioPak) or 1xTE.
- There is widespread belief that RNA is very unstable and therefore all the reagents and materials for its handling should be specially treated to remove possible RNAse activity. We have found that purified RNA is rather stable and, ironically, too much anti-RNAse treatment can become a source of problems. This especially applies to DEPC-treating of aqueous solutions, which often leads to RNA preparations that are very stable but completely unsuitable for cDNA synthesis. We have found that simple precautions such as wearing gloves (only for your protection from chemicals), avoiding speech over open tubes, using aerosol-barrier tips, and using fresh 1xTE (or 1xTHE) solution (or Milli-Q ultrapure BioPak water) for all solutions are sufficient to obtain stable RNA preparations.
When an ultrafiltration cartridge (BioPak) is utilized at the point-of-use, the water is suitable for genomics applications (quality at least equivalent to DEPC-treated water) and cell culture. The BioPak cartridges has been validated in Millipore laboratories to warrant the production of pyrogen-free (less than 0.001 Eu/ml), RNAse-free (less than 0.01 ng/ml) and DNase-free (less than 4 pg/μl) ultrapure water, while maintaining both the resistivity and total organic carbon (TOC) of the treated water, it replaces the lengthy diethylpyrocarbonate (DEPC) treatment process to remove nucleases from purified water.
All organic liquids (phenol, chloroform and ethanol) can be considered essentially RNAse free by definition, as is the dispersion buffer containing guanidine thiocyanate. The volume of tissue should not exceed 1/5 of the extractiom buffer volume. To avoid RNA degradation, tissue dispersion should be carried out as quickly and completely as possible, ensuring that cells do not die slowly on their own. To adequately disperse a piece of tissue usually takes 2-3 minutes of triturating using a pipet, taking all or nearly all volume of buffer into the tip each time. The piece being dissolved must go up and down the tip, so it is sometimes helpful to cut the tip to increase the diameter of the opening for larger tissue pieces. Tissue dispersion can be performed at room temperature. The tissue dispersed in extraction buffer produces a highly viscous solution. The viscosity is usually due to genomic DNA. This normally has no effect on the RNA isolation (except for dictating longer periods of spinning at the phenol-chloroform extraction steps), unless the amount of dissolved tissue was indeed too great. RNA degradation can be assessed using non-denaturing electrophoresis. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands do not have a discernible lower edge, the RNA preparation should be discarded. The amount of RNA can be roughly estimated from the intensity of the rRNA staining by ethidium bromide in the gel, assuming that the dye incorporation efficiency is the same as for DNA (the ribosomal RNA may be considered a double-stranded molecule due to its extensive secondary structure). The rule for vertebrate rRNA - that in intact total RNA the upper (28S) rRNA band should be twice as intense as the lower (18S) band - does not apply to invertebrates. The overwhelming majority have 28S rRNA with a so-called "hidden break". It is actually a true break right in the middle of the 28S rRNA molecule, which is called hidden because under non-denaturing conditions the rRNA molecule is held in one piece by the hydrogen bonding between its secondary structure elements. The two halves, should they separate, are each equivalent in electrophoretic mobility to 18S rRNA. In some organisms the interaction between the halves is rather weak, so the total RNA preparation exhibits a single 18S-like rRNA band even on non-denaturing gel. In others the 28S rRNA is more robust, so it is still visible as a second band, but it rarely has twice the intensity of the lower one.
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Current and disassemble the approval of the oxygen atom and add molecular and ethanol precipitation respectively were performed a portion
RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.
The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues.TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. RNA remains only in the aqueous phase.
After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But the DNA and proteins can recover by sequential separation after the removal of aqueous phase. Precipitation with ethanol requires DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay, and molecular cloning.
- The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes.
- 0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL LS Reagent was added. The tubes were shaked vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes.
- The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C).
- The aqueous phase was transferred to other tubes. ( Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL LS Reagent used for homogenization.)
- The RNA was precipitated from the aqueous phase by mixing with 3 microlitre of glycogen and 500 microlitre of isopropyl alcohol.
- The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C).( The RNA precipitate forms a gel-like pellet on the side of the tube at bottom).
5. Assessing the Validity of Crosslinking Data
Determining the structural relevance of a crosslink should include consideration of the following criteria. First, the number and distribution of observed crosslinks should be consistent with that normally observed with a given crosslinking agent. Crosslinking with long-range structure probes such as APA usually involves several adjacent nucleotides in 1𠄴 distinct regions of the target RNA while the number of nucleotides and crosslinked regions of RNA is significantly reduced in short-range structural probes such as 6sG and 4sU. The general suspicion of a structurally heterogeneous population of RNA should thus be raised when the number or distribution of observed crosslinks exceeds the general guidelines noted above. This should be initially be addressed by a re-examination of renatur-ing conditions prior to the crosslinking reaction, followed changes in the placement of the crosslinking reagent itself.
Second, the efficiency of crosslinking provides correlative rather than direct evidence for structural proximity or conformational stability. The strong geometrical and chemical requirements for bond formation dictate that the relative proximity of two crosslinked sites is not strictly linked to the level of crosslinking that is actually observed. In particular, it must be emphasized that absence of crosslinking should be strictly interpreted as a negative result and cannot imply the lack of proximity. Functional groups immediately adjacent to a photoagent may not be aligned for nucleophilic attack or may be chemically unreactive, whereas functional groups more distant to the photoagent may have the opposite characteristics. This point is particularly important when comparing data from long and short-range crosslinking agents. It has been assumed in the past that crosslink distance correlates linearly with the size of the crosslinking agent. However, this correlation was not observed when long and short-range crosslinking studies were compared in the context of established crystallographic structures (Sergiev et al., 2001 Whirl-Carrillo et al., 2002). While the absence of correlation may be partially due to experimental error (e.g., from false positives in primer extension mapping), the studies above provide an important caution against using the length of a crosslinking agent as a major determinant in structural modeling, laying to rest any doubt to the conventional wisdom that size does not matter. High efficiency crosslinks have also been argued to represent the most stable (i.e., native) structure in the population. Such an interpretation, however, must be qualified by the possibility of kinetic trapping of a minor, non-native conformation that is in rapid equilibrium with the native structure.
Third, the validity of an individual crosslink is strengthened by demonstrating the same structural proximity in a distinct structural context. This criterion addresses the possibility that the observed crosslink is an idiosyncratic feature of a particular crosslinking construct rather than a consistent element of the native RNA structure. The most direct approach to addressing this concern is to determine whether the same nucleotides or regions of RNA structure become crosslinked regardless of which of the nucleotides or regions of RNA in question contains the crosslinking agent (Chen et al., 1998 Harris et al., 1997). The demonstration of reciprocal crosslinks from different photoagents or under different experimental conditions provides further support that the observed results are not due to the perturbation of the native structure. Generality of the crosslinking results can also be established by reproducing the crosslink in a homologous RNA (Chen et al., 1998 Christian et al., 1998 Harris et al., 1994, 1997 Noah et al., 2000). Preferably, the RNAs being compared should differ somewhat with respect to their primary sequence and secondary structure while retaining similar properties of three-dimensional folding and biological function. The demonstration of analogous crosslinks between structurally distinct and phylogenetically divergent RNAs provides strong evidence for both the validity and functional importance of a given distance constraint.
Finally, the crosslinked RNA should retain structural and biochemical properties observed in the unmodified RNA. Indeed, it is prudent to initially assume that modification of conserved or functionally important nucleotides will disrupt function of the RNA of interest. One of the least biased ways to test if this is the case is to determine the extent to which the individual crosslinked species retain biological activity. Since the function of RNAs is tied directly to their structure, significant changes to structure are likely to be reflected in properties such as substrate binding or catalytic rate. Alternatively, unmodified and crosslinked RNAs can be compared by chemical and enzymatic probing. Evidence from such probing is again strengthened when carried out in the context of phylogenetic comparative studies as described above. Ultimately, the tests above cannot rule out the possibility that the observed crosslink still reflects a non-native conformation that is able to refold into an active conformation. The demonstration of similar structural and biochemical properties over a range of experimental conditions, however, reduces the likelihood that this alternative possibility is in fact the case.