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View additional product information for TRIzol™ Reagent - FAQs (15596018CN)
64 product FAQs found
Degraded RNA can cause an increased absorbance at 260 nm.
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This is most likely polysaccharides or cell membranes; DNA should be in the interphase. In samples containing blood (e.g., liver), a red viscous layer may be visible on top of the pellet. This is most likely due to blood products and should not be carried over with the supernatant.
- This is common with skin samples. It is assumed that there is fat in these samples, and the fat micelles float during the centrifugation. In skin samples, the micelles pick up melanin pigment and cause the aqueous phase to appear colored. Fat micelles may also pick up pigment from the TRIzol Reagent itself and cause a pinkish color. If a sample is thought to contain fat, the sample homogenate in TRIzol Reagent may be centrifuged prior to addition of chloroform. The fat will appear as a clear layer at the top of the supernatant; this should be pipetted off and discarded.
- If a sample contains a lot of blood, the aqueous phase may appear cloudy and/or yellowish (this may be due to iron in the hemoglobin). If the centrifuge used is not cold, the organic phase will be a deeper maroon color; some of this color may come into the aqueous phase and cause it to appear orange or yellow.
- A pinkish aqueous phase may also be caused by overdilution of the sample (i.e., a sample to TRIzol Reagent ratio > 1:10), as well as too much salt or protein in the sample. This can cause premature phase separation, which can be remedied by adding a bit more TRIzol Reagent to the sample. If the RNA is isolated from a pinkish aqueous phase, chances are that it will be contaminated with DNA.
These are our recommendations:
1. Upstream tissue procurement and tissue specimen preparation—if possible, fix tissues within one hour of surgical resection. Extensive degradation of RNA can occur before completion of the fixation process. The optimal fixation time is 12-24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
2. Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungus, insects, etc.).
3. Choice of tissue type, size, and amount being used for RNA isolation—the recommended tissue thickness is 10-20 µm. The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50-300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
4. Avoid using an excessive amount of paraffin for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, you can perform a more rigorous 37-55 degrees C treatment for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.
Read more about RNA isolation from FFPE tissues here (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-sample-extraction/working-with-ffpe-samples.html).
If a sample is known to have a high content of proteoglycans and/or polysaccharides (such as rat liver, rat aorta, plants), the following modification of the RNA precipitation step should remove these contaminating compounds from the isolated RNA:
- Add 0.25 mL of isopropanol to the aqueous phase followed by 0.25 mL of a high-salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl; no pH adjustment necessary) per 1 mL of TRIzol Reagent used for homogenization. Mix the resulting solution, centrifuge, and proceed with isolation as described in the protocol.
This modified precipitation effectively precipitates RNA and maintains proteoglycans and polysaccharides in a soluble form. To isolate pure RNA from plant material containing a very high level of polysaccharides, the modified precipitation should be combined with an additional centrifugation of the initial homogenate. In general, we do not recommend high-salt precipitation if polysaccharide or proteoglycan contamination is not a concern, since it is an extra step and there is otherwise no significant advantage to adding this step. When purifying an RNA sample where polysaccharide or proteoglycan contamination is not an issue, in general, the total RNA yield will be same with or without the high salt. There may be small changes in the RNA profile reflected by slightly decreased amounts of tRNA. The high-salt precipitation reduces tRNA in the sample.
You can add glycogen to your sample, which can help improve yield and remains with the RNA (glycogen is water soluble). Polyacrylamide can also be used as a carrier to precipitate small amounts of RNA. Alternatively, you can also use salmon sperm DNA. It should be added during the precipitation of the aqueous phase.
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- The sample was homogenized in too small a volume of TRIzol Reagent.
- The sample was not stored at room temperature for 5 minutes after homogenization. (This may result in nuclear proteins not being dissociated).
- The final RNA pellet was not fully dissolved. This may be the case if the RNA pellet was overdried (if the pellet is clear and not white, this indicates overdrying). To get the pellet to dissolve completely, heat to 55-60 degrees C for 10-15 minutes and repeatedly pipette.
- There may be phenol contamination. This may occur if samples were centrifuged at room temperature instead of 4 degrees C; phenol is more soluble in the aqueous phase at room temperature. If absorbance is seen at 270 nm (due to phenol), the sample can be ethanol precipitated to remove the residual phenol.
- Guanidine absorbs around 240 nm. Phenol has two peaks: one around 275 nm, the other a broad peak ranging from below 220 to around 240 nm. If a very large peak is observed in that range, we recommend that you precipitate and wash again. To prevent this, we also recommend doing the phase separation after addition of chloroform at 4 degrees C.
- Residual chloroform may be present; reprecipitate.
- In some samples dissolved in water, the ratio may be low due to the acidity of the water or the low ion content in the water. The ratios may go up if the sample is dissolved in TE and the spectrophotometer is zeroed with TE buffer (or 1-3 mM Na2HPO4, pH approximately 8.0). The molar extinction coefficient of the nucleotides is given at neutral pH, suggesting that the absorbance at 260 nm would be highest at neutral pH.
- A variation in A260/A280 ratios with different spectrophotometers (it seems that the A280 value varies depending on the way the unit was “blanked” at 280 nm after being “blanked” at 260 nm). In this case, we suggest using a different spectrophotometer.
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A portion of the interphase may have been removed with the aqueous phase after the initial separation. This can occur for several reasons.
- An insufficient amount of TRIzol Reagent was added to the sample. In general, 1 mL of TRIzol Reagent should be used for every 0.05 g of tissue or every 10 cm2 dish.
- The original sample may have had traces of other organic material to begin with (ethanol, DMSO, etc.).
- RNA should be treated with amplification grade DNase I prior to RT-PCR.
- There may have been insolubles after the first homogenization that were not removed by centrifugation before chloroform extraction.
- If the DNA contamination was detected in RNA isolated from cells after transfection with a plasmid, not all of the plasmid DNA may have partitioned into the interphase/organic phase once the chloroform was added to the TRIzol Reagent. If RT-PCR is being used to assay gene expression from the transfected plasmid, a DNase I treatment will be needed.
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Please review the following causes for low yield of RNA/degraded RNA:
- The RNA may have been concentrated with a SpeedVac system or lyophilized after the last ethanol precipitation. RNA that has been dried completely has decreased solubility. Additionally, if excess centrifugation speeds (higher than 12,000 x g) were used, it is harder to solubilize RNA/DNA.
- The RNA pellet may not be completely solubilized. To increase the rate of solubilization, pipette repeatedly in SDS solution or DEPC-treated water, then heat to 50-60 degrees C. The sample may also have been rich in polysaccharides or proteoglycans. If so, the isopropanol precipitation step should be done with 0.25 volumes of isopropanol and 0.25 volumes of a high salt solution.
- Cells were washed prior to the addition of TRIzol Reagent. Washing cells before the addition of TRIzol Reagent increases the possibility of mRNA degradation.
- The sample was not fully homogenized.
- The tissue was not IMMEDIATELY processed or frozen after removal from the animal or other source.
- The tissue was not completely disrupted; if a centrifugation is done prior to adding chloroform, there should be a white mucus-like pellet. If there is a tan-colored precipitate, this is indicative that not all of the cells have been lysed.
- If a mortar and pestle was used to powder the tissue, RNA and DNA may have stuck nonspecifically to the mortar and pestle. It may be better to use a glass homogenizer and Teflon pestle; add TRIzol Reagent to the homogenizer, then add frozen tissue and homogenize.
- RNA may have been stored after isolation at -20 degrees C instead of -70 degrees C.
- Tissue culture cells were disrupted by trypsin.
- Homogenizing for too long and too continuously in a small volume (e.g., 1 mL) may cause heating of the sample; this may result in degradation of the RNA in the tissue. Samples should be cooled during homogenization, and homogenization should be done in on-off cycles (as opposed to continuously).
- The OD reading may vary due to the solution the sample is stored in AND what it was diluted in prior to quantitation. This can lead to apparently low yields.
- Excess RNAlater Stabilization Solution (>0.05 mL) will reduce RNA recovery and cause problems with phase separation.
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There are two methods to remove insoluble material:
1. For RNA isolation only: If a lot of insoluble material exists after homogenization and a 5 minute room temperature incubation, remove it by centrifugation at 12,000 x g for 10 minutes at 4 degrees C before adding chloroform (a clear supernatant and jelly-like pellet should be seen). Remove the supernatant and proceed to the next step. Note: This should not be done if subsequent DNA isolation is planned.
2. RNA and DNA isolation: If a lot of insoluble material exists after homogenization and a 5 minute room temperature incubation, the homogenate can be passed through a polypropylene mesh to remove insoluble material that may interfere with precipitation of DNA.
Please visit our website (http://www.thermofisher.com/content/dam/LifeTech/migration/en/images/ics-organized/applications/nucleic-acid-purification/data-image/560-wide.par.83692.image.559.294.1.gif) for a graph showing purity measurements and RNA integrity number (RIN) comparison of the abovementioned kits.
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The TRIzol Plus RNA Purification Kit combines the lysis capability of TRIzol Reagent with the convenient RNA extraction technology of the silica spin columns included in the PureLink RNA Mini Kit.
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Yes, you can use TRIzol Reagent or the mirVana PARIS RNA and Native Protein Purification Kit.
If the aqueous phase was removed completely and ethanol was added to the samples, it will remain on top of the TRIzol Reagent due to ethanol's lower density. If the samples were centrifuged without mixing the two liquids, the ethanol will remain on top of the TRIzol Reagent after centrifugation, the DNA will remain at the interface, and the TRIzol Reagent will be localized to the red organic fraction on the bottom. If the ethanol was not mixed properly, proceed with mixing the samples, then centrifuge and continue to step 1 of the DNA isolation protocol.
If 70% ethanol was added accidentally, it may be possible to get a small volume of water on top of the organic fraction. Since the wash solutions that are used in the protocol do not exceed 30% water, you would expect to see no more than 30% of 0.3 mL (90 µL) of water on top of the organic fraction. You can try removing and discarding the water before proceeding with the isolation. DNA yield may be decreased.
This could also happen if the phase separation was not complete during the RNA isolation step. This can occur because the chloroform was not adequately mixed or if the samples were not centrifuged at the proper g-force or for the required period of time or at the correct temperature. The net result is that significantly less than 600 µL of the RNA aqueous phase will be recovered from the sample. Phase separation problems usually occur when the chloroform is mixed in the tube by vortexing. Due to the large difference in density between TRIzol Reagent and the organic phase, the solutions are never mixed completely and only a portion of the aqueous phase will be recovered. When the ethanol is added and the samples are remixed sufficiently, the phase separation will go to completion and water could appear on top of the sample.
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Typically, low absorbance is due to phenol contamination, which can occur if samples were centrifuged at room temperature instead of 4 degrees C. We recommend a second ethanol precipitation to remove remaining phenol.
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Here are some possible causes for low yield/DNA degradation:
- The sample was not fully homogenized or lysed. If any solid material remains after chloroform is added, this indicates that DNA yield may be poor, as DNA will remain trapped in the unhomogenized material.
- The final DNA pellet was not fully redissolved, so please make sure that 8 mM NaOH was used as reuspension solution. If the DNA is not fully redissolved, it will be lost during the final centrifugation when removing the gel-like material.
- The tissue was not immediately processed or frozen after removal from the animal or other source.
- Samples were homogenized with a high-speed homogenizer. DNA shearing can happen.
- If expected yield is <10 µg, there are limitations to the physical action of precipitation that would lead to low yields. A microcarrier (glycogen, tRNA) may be included in the homogenization and/or wash steps, or samples may be pooled to increase the expected yield.
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The most common problem related to DNA solubilization occurs when the DNA pellets are overdried. It is very important not to dry pellets longer than 10 minutes. If you follow our simple recommendations below, you can avoid many nucleic acid solubility problems.
Remove droplets of ethanol from the wall of the test tube with a sterile cotton swab. Additional ethanol can be removed by touching the pellet with a sterile capillary pipette tip. Excess ethanol will be drawn inside the pipette by capillarity. Residual ethanol that may remain in the DNA pellet will not be harmful. You can usually eliminate DNA solubility issues by adding 8 mM NaOH to the pellet before all of the ethanol has evaporated. The DNA pellets will become clear after a 5-10 minute incubation, as they begin to rehydrate. In order to solubilize the DNA completely, the solution must be pipetted up and down before removing an aliquot for quantitation.
DNA pellets that are overdried can be solubilized but it may be necessary to put them into the refrigerator and pipet them periodically until they become clear and go into solution.
Polysaccharides are water-soluble and they will partition into the aqueous phase with the RNA. Also, RNA and DNA pellets that contain contaminants tend to solubilize more easily than pellets that are very pure if they are not overdried. Pellets that do not solubilize in 8 mM NaOH will not solubilize in a phenol/chloroform solution, either.
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Yes, single-stranded DNA will separate with the DNA phase.
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The phenol phase and interphase can be stored at 4 degrees C overnight. Samples can also be stored in the washing solution (0.1 M sodium citrate in 10% ethanol) for at least a couple hours. The samples can also be suspended in 75% ethanol at 4 degrees C for several months.
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Yes, proteins can be precipitated by the addition of isopropanol or acetone. Optimal protein yield can be achieved with an acetone:phenol-ethanol ratio between 3:1 and 6:1.
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A low ratio can be caused by several factors:
- Sample homogenized in too small a volume of TRIzol reagent.
- Samples not stored at room temperature for 5 minutes after homogenization. This may result in nuclear proteins not being dissociated.
- Final RNA pellet was not fully dissolved. This may be the case if the RNA pellet was overdried (if the pellet is clear and not white, this indicates overdrying). To get the pellet to dissolve completely, heat to 55 to 60 degrees C for 10–15 minutes and repeatedly pipet.
- Phenol contamination (this may occur if samples were centrifuged at room temperature instead of 4 degrees C; phenol is more soluble in the aqueous phase at room temperature). If absorbance is seen at 270 nm (phenol), sample can be ethanol precipitated to remove residual phenol.
- Residual chloroform is present; re-precipitate your sample.
- OD reading may vary with sample storage solution and diluant.
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TRIzol Plus reagent combines TRIzol reagent and a column-based purification system in order to isolate the highest quality RNA. The column purification helps to remove 18s RNA and tRNA, removes any trace of phenol, and provides for double gDNA removal.
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The following suggestions may be useful for the precipitation of small RNA species (<250 bp):
Increase the amount of isopropanol used to precipitate from 0.5 up to 1.0 mL (per mL TRIzol reagent). This will improve the recovery
of small molecular weight RNA. It may also increase salt contamination, so the volume of isopropanol that is required to
efficiently precipitate this RNA without increasing the salt precipitate from the aqueous phase will have to be
determined in each case.
Perform the precipitation step in the absence of tissue to observe the degree of salt precipitation
and assess the proper amount of isopropanol to use.
Monitor A230 to determine if salt precipitation is increasing. The A260:A230 ratio should be greater than 1.7.
If some salt is precipitating, it may be possible to remove it by adding a second wash step with 75% ethanol.
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If a sample is known to have a high content of proteoglycans and/or polysaccharides (such as rat liver, rat aorta, plants), the
following modification of the RNA precipitation step should remove these contaminating compounds from the isolated RNA:
Add 0.25 mL of isopropanol to the aqueous phase followed by 0.25 mL of a high salt precipitation solution (0.8 M sodium
citrate and 1.2 M NaCl (no pH adjustment necessary)) per 1 mL of TRIzol reagent used for homogenization.
Mix the resulting
solution, centrifuge, and proceed with isolation as described in the protocol.
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For long-term storage, we recommend resuspending the RNA in stabilized formamide and storing at -70 degrees C. To remove the formamide, add 4 volumes ethanol and, if less than 20 µg RNA, also add NaCl to 0.2 M. Precipitate the RNA and use for downstream experiments.
Yes, you can use salmon sperm DNA as a carrier. Add it during the precipitation of the aqueous phase.
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Yes, centrifugation speeds as low as 5,000 to 6,000 x g have been used, but the centrifugation time should be doubled to get the expected yields.
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If a large amount of chloroform was inadvertently added, you can add more TRIzol Reagent so that the ratio of 0.2 mL chloroform:1 mL TRIzol Reagent is maintained. If too much chloroform is added, this can drive the DNA, and eventually the protein, into the aqueous phase.
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If isopropanol is inadvertently added at this step instead of chloroform, add more isopropanol to precipitate everything, then resuspend the pellet in TRIzol Reagent and use the protocol as specified. RNA yields will be compromised, but it may be possible to obtain a product in RT-PCR. A detailed protocol follows:
(1) Add more isopropanol so that the total volume of isopropanol equals the volume of TRIzol Reagent used. Spin at 7500 x g for 10 min at 4 degrees C.
(2) Pour off supernatant; allow relatively compacted pellet to air dry (doesn't have to be completely dry, just reduce the volume of ispropanol).
(3) Estimate the size of the pellet in microliters; add at least 15–20 volumes of TRIzol Reagent (e.g., for a 100 µL pellet, add at least 1.5 mL TRIzol Reagent).
(4) Break the pellet up well (you may have to use a hand-held homogenizer). Store the solution for 10–15 min. at room temperature; every 5 min or so, shake it by hand to make certain it is well dispersed.
(5) Proceed with the TRIzol Reagent protocol as written (i.e., add chloroform). Results will not be optimal, but it may be possible to get a product in RT-PCR.
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Glycogen can be included with your sample to improve yield, and remains with the RNA (glycogen is water soluble). Polyacrylamide can also be used as a carrier to precipitate small amounts of RNA.
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There are a couple of possible stopping points in the RNA extraction protocol as shown below:
•After homogenization (before addition of chloroform), samples can be stored at 4 degrees C overnight or at –70 degrees C for at least 1 year.
•Homogenized samples can sit at room temperature for several hours before chloroform is added.
•Homogenized samples can be thawed and refrozen prior to use (necessary when researcher intends to do experiment, but then cannot continue).
• After RNA precipitation, during RNA wash, the RNA can be stored in 75% ethanol for at least 1 year at –20 degreesC, or at least 1 week at 4 degrees C.
For DNA extraction, the phenol phase and interphase can be stored at 4 degrees C overnight before DNA precipitation. Some customers have tried storing at 4 degrees C for a week and –20 degrees C for a year and still got good recovery.
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You can homogenize your sample using a glass Teflon or power homogenzier (Polytron or Tekmar's Tissumizer) in a 1.5 microcentrifuge tube. Cultured cells do not have to be homogenized. Sonication will work to lyse cells in TRIzol reagent, but should only be performed if you do not plan on isolating DNA from your sample. Cells grown in monolayers can be lysed directly in the culture dish.
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Yes, tissue stored in RNAlater Reagent can be used in the TRIzol Reagent. Remove the tissue from RNAlater Reagent, and immediately submerge in TRIzol solution.
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TRIzol LS Reagent is a more concentrated formula, allowing for lower quantities of the reagent to be used relative to the sample. LS stands for liquid samples. TRIzol LS Reagent is formulated with a reduced volume of water to allow addition of a larger sample volume to a smaller volume of reagent. Therefore, when TRIzol Reagent and TRIzol LS Reagent are used in accordance with their respective protocols, they will perform identically and contain the same amount of chaotropic agents. The two reagents can be distinguisehd by color, where TRIzol LS Reagent is a darker, maroon red while TRIzol Reagent is lighter in color.
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The TRI stands for Total RNA Isolation. It also signifies the fact that this reagent can be used in the purification of RNA, DNA, and proteins from a single source.
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TRIzol Reagent is a ready-to-use mixture of phenol, guanidine isothiocyanate, red dye, and other proprietary components.
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Yes you can. Here is a reference to a paper, as well as a brief description of method from the paper.
Genes to Cells (2001) 6:121-129 (under the heading 'RNA isolation and RT-PCR')
TRIzol LS Reagent (LifeSciences) was used according to the manufacturer's instructions to extract total RNA from sucrose gradient fractions. Briefly, 250 mL of each fraction was added to 750 mL TRIzol LS Reagent and shaken vigorously for 15 s. After a 10-min incubation at room temperature, 150 mL chloroform was added, followed by vigorous shaking and brief incubation at room temperature. Samples were then spun at 14,000 g for 10 min in a tabletop microcentrifuge. Five micrograms of nuclease-free glycogen were added to 300 mL of the aqueous phase and nucleic acids were precipitated with the addition of an equal volume of 2-propanol. After centrifugation at 14,000 g for 30 min at room temperature, the pellet was washed once with 75% ethanol and resuspended in 20 mL of nuclease-free, sterile water. Five microlitres of total RNA were used as substrate for random-primed cDNA synthesis using Superscript II modified MMLV reverse transcriptase (Gibco/Life Sciences).
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TRIzol Reagent contains phenol and guanidine isothiocyanate, which allow for isolation of nucleic acids from proteins. Further partitioning of the nucleic acids occurs in a pH dependent manner. At pH 7.0 or higher, DNA and RNA partition into the aqueous phase. At an acidic pH, below 7.0, DNA is denatured and will move into the organic phase (interphase) yet the RNA remains in the aqueous phase.TRIzol Reagent has pH about 5.
Blood collected with EDTA typically has the highest DNA contamination, blood collected with heparin typically has less than that collected with EDTA, and blood collected with citrate shows the least DNA contamination of the three. (Formulation for citrate solution: 3.8% (w/v) which is 3.8 g/100 mL of water. Use 0.5 mL for every 4.5 mL of blood. Rock gently back and forth after adding citrate solution to mix.) Adding 12 µL of 5 N acetic acid per milliliter of TRIzol Reagent may help, although there may still be a problem with DNA contamination. Using plasma or serum works best. The fresher the blood sample the better the RNA. Degraded RNA has been observed in blood that has been processed in as little as two hours after drawing.
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There are a couple of reasons that the aqueous phase may appear pinkish. It may be due to the tissue you are using. This is common with skin samples. It is assumed that there is fat in these samples, and the fat micelles “try to spin to the top of the tube” during the centrifugation but are unable to get there. In skin samples, the micelles pick up melanin pigment and cause the aqueous phase to appear colored. Fat micelles may also pick up pigment from the TRIzol Reagent itself, resulting in a pinkish color. If a sample is thought to contain fat, the sample homogenate in TRIzol Reagent may be centrifuged prior to addition of chloroform. The fat will appear as a clear layer at the top of the supernatant; this should be pipetted off and discarded.
In addition, if a sample contains a lot of blood, the aqueous phase may appear cloudy and/or yellowish (this may be due to iron in the hemoglobin coming out).
If the centrifuge used is not cold, the organic phase will be a deeper maroon color; some of this color may come into the aqueous phase and cause it to appear orange or yellow.
Alternatively, a pinkish aqueous phase may also be caused by over-dilution of the sample (i.e., the sample:TRIzol Reagent ratio which is greater than 1:10), as well as too much salt or protein in the sample. This can cause premature phase separation, which can be remedied by adding a bit more TRIzol Reagent to the sample. If the RNA is isolated from a pinkish aqueous phase, chances are that it will be contaminated with DNA. Although this should not be significant as TRIzol Reagent is formulated to prevent premature phase separation.
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TRIzol LS Reagent is a complete, ready-to use reagent for easy and simultaneous isolation of total RNA, DNA, and proteins from liquid samples. The reagent, a mono-phasic solution of phenol and guanidinium thiocyanate, is an improvement to the single-step RNA isolation method developed by Chomzcynski and Sacchi. TRIzol LS Reagent is similar to the original TRIzol Reagent in composition and results of use. TRIzol LS Reagent is designed for use with liquid samples such as blood and virus preparations in which large volumes of aqueous samples need to be processed, while TRIzol Reagent is designed for cell cultures or tissues. It is formulated to accommodate processing of more liquid sample per unit of reagent compared to the original formula.
The only difference between TRIzol Reagent and TRIzol LS Reagent is the concentration of components. TRIzol LS Reagent is slightly more concentrated. The formula allows lower quantities of reagent to be used relative to a liquid sample. (TRIzol = 10:1 required, TRIzol LS = 3:1 required). If you buy TRIzol LS Reagent, but want to use it like TRIzol Reagent (on solid samples), there will probably be a decrease in yield vs. using regular TRIzol Reagent. TRIzol LS Reagent should NOT be used undiluted with solid samples. To dilute: take 750 µL TRIzol LS Reagent + (50 to 100 mg tissue + water to make 250 µL).
Our TRIzol Reagent (Cat. No. 15596026) can isolate RNA, DNA, and protein from the same sample.
We recommend the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975). This kit is optimized for isolation of both DNA and RNA from formalin or paraformalin-fixed, paraffin-embedded (FFPE).
Another option is TRIzol Reagent, but be sure to check the references listed below. Because paraffin is not soluble in TRIzol Reagent, paraffin-embedded tissues can be quick-heated to get the tissue out of the paraffin; any paraffin which remains will float to the top of the aqueous phase (and should be avoided). (If the slice is very thin, the whole slice can be added to the TRIzol Reagent, and hopefully, the tissue will be exposed to the reagent). Most of the references we surveyed do not provide quantitative data, because paraffin-embedded tissues are dramatically influenced by the action of nucleases prior to fixation and by the formalin fixation time.
The ability to detect specific housekeeping genes by PCR analysis with RNA or DNA extracted from these tissues is usually considered to be a positive result. We do not have a protocol per se, but we have spoken with customers who are doing this. We recommend deparaffinizing with xylene (or other organic), then grinding the sample very thoroughly in TRIzol Reagent (may require a Polytron); in most cases, you have to homogenize with vigor because the DNA is crosslinked and you have to get it free. Microcarrier is recommended since the RNA is crosslinked and fragmented. From this point, the standard isolation protocol can be used. They have found publications that show that the success of the isolation is dependent on how long the sample was fixed (there is an inverse relationship): Inoue, T., et. al., Pathology International (1996) Vol 46, Iss 12, pp. 997-1004.
If you will not need to isolate genomic DNA from the same sample and want to reduce the chance of gDNA contamination in your RNA, you should perform the optional centrifuge step mentioned in step 1 of the TRIzol Reagent manual prior to addition of chloroform.
After homogenizing your sample thoroughly in TRIzol Reagent, centrifuge the sample at 12,000 X g for 10 minutes at 4 degrees C. Genomic DNA, cellular membranes, and polysaccharides will form a pellet, and your RNA will be in the supernatant. Any lipids and fats in your sample may form a layer at the top of the solution as well. Remove the fat layer if necessary with a sterile tool and transfer the RNA supernatant to a new vial. Discard the DNA pellet.
Add chloroform to the RNA supernatant and proceed with the RNA isolation protocol.
To reduce gDNA contamination even more, you can treat your RNA after isolation with amplification grade DNase I. (Using non-amplification grade DNase I is not recommended, as it is not validated for absence of RNases and has been shown to degrade RNA samples in some cases.)
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Add 10 micrograms of RNase-free glycogen to less than 10 mg tissue or less than 1 X 10e6 suspension cells. Glycogen, unlike salmon sperm DNA carrier, can be added when TRIzol Reagent is added to sample.
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Pellet polysaccharides (also pellets genomic DNA): Centrifuge following homogenization before adding chloroform at 12,000 X g at 4 degrees C for 10 min to pellet polysaccharides. In addition, you may need to do a high-salt isopropanol precipitation as follows.
After collecting the aqueous phase, add 0.25 mL isopropanol and 0.25 mL of 0.8 M sodium citrate, 1.2 M NaCl per 1 ml TRIzol Reagent. Mix the solution, centrifuge, and proceed with isolation as described. This precipitates the RNA and maintains proteoglycans and polysaccharides in a soluble form. Samples known to have a high content of proteoglycans or polysaccharides include rat liver, rat aorta, and plants.
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Yes. We have been successful in-house with 104 cells using 0.4 ml of TRIzol Reagent. When precipitating the RNA, add 5-10 µg of RNAse-free glycogen as a carrier to the aqueous phase.
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Yes. Scale up linearly for tissues and suspension cells. For monolayer cells, scale up linearly based on the surface area of the plate, NOT the amount of cells. Use at least 1 ml of TRIzol Reagent for every 10 cm2 of surface area.
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RNA dissolved in deionized formamide can be stored at -70 degrees C for up to 1 year. To precipitate RNA from formamide, add NaCl to final concentration of 0.2 M followed by 4 volumes of ethanol. Incubate 3-5 min at room temperature and centrifuge at 10,000 x g for 5 min.
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Use polypropylene tubes. Do not use tubes sensitive to phenol.
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Yes, Use 2,600 X g at 4 degrees C for 60 min for phase separation and 30 min for RNA precipitation.
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About 60% of the TRIzol Reagent volume becomes part of the aqueous phase after chloroform addition.
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The DNA pellet from precipitation with isopropanol is easily dislodged when washing with 70% ethanol. It is best to remove the isopropanol supernatant and the ethanol wash by pipetting. Be careful not to shoot the washing buffer directly onto the pellet. Instead, allow the washing buffer to run over the pellet. Regardless of which manufacturer's miniprep kit you use, washing the pellet can be challenging because it is so small.
Yes. However, since the DNA is dissolved in 8 mM NaOH, the pH needs to be adjusted with HEPES. Please contact Technical Service for details. Depending on the restriction endonuclease, use 3 to 6 units per µg of DNA for between 3 hr and overnight. In a typical assay, 80 to 90% of the DNA is digestible.
There are already components of the TRIzol Reagent that function as anti-foaming agents. The addition of other anti-foaming agents has not been tested. If you attempt adding other components, you may experience higher DNA contamination. If you are working with very small samples, we might suggest using a slightly larger volume of TRIzol Reagent in order to minimize loss of sample when homogenizing. Of course, as always, use caution when adding anything to an RNA purification reagent as RNA degradation is always a concern.
There are several Thermo Fisher Scientific products that can be used to purify RNA from plants. They are listed in alphabetical order:
Plant RNA Isolation Reagent.
--Plant RNA reagent is designed to isolate high yields of RNA from plant samples, even difficult ones such as conifer and plant seeds. This reagent does a good job of removing phenolics and starches.
TRIzol and TRIzol LS reagents.
--TRIzol Reagent is a good choice unless the tissue is very watery (such as fruit), in which case TRIzol LS Reagent would be recommended. When using TRIzol Reagent with plant tissue, we recommend that you homogenize 15 mg (total weight) leaf with ground glass homogenizer in 1 mL of TRIzol Reagent. Proceed with the rest of the protocol. In poinsettia, we have observed 33 µg RNA from 45 mg tissue (0.7 µg/mL) and from tobacco 76 µg RNA from 100 mg tissue (0.8 µg/mL).
Special considerations for RNA precipitation from tissue containing high amounts of proteoglycans and/or polysaccharides (as found in plants): Add to the aqueous phase 0.25 mL of isopropanol followed by 0.25 mL of a high-salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl) per 1 mL of TRIzol Reagent used for homogenization. Mix the resulting solution, centrifuge, and proceed with isolation as described in the protocol. The modified precipitation effectively precipitates RNA and maintains proteoglycans and polysaccharides in a soluble form. This procedure should ONLY be used if the sample is known to have a high content of proteoglycans and polysaccharides. To isolate pure RNA from plant material containing a very high level of polysaccharides, the modified precipitation should be combined with an additional centrifugation of the initial homogenate.
Special considerations for RNA precipitation from cotton leaf: Yields from 100 mg tissue were almost non-existent. We have noted that DNA yield from cotton leaf is 1/6 to 1/3 that of other plants, so there may be a correlation with RNA yield as well. Try using 3 to 5X more tissue and a microcarrier, as well as the pre-spin (before chloroform) and modified (high salt) precipitation. Also, some plants when homogenized in TRIzol Reagent will change the pH; it should be 5.5 (measuring with pH paper is accurate enough). If it is higher, adjust using glacial acetic acid.
TRIzol Reagent has demonstrated stability of 12 months when stored at room temperature. However, we recommend storage at 2 to 8 degrees C for optimal performance.
No, the stability of TRIzol Reagent will not be impacted by storing it at room temperature overnight. The 4 degrees C storage condition for the reagent helps with ease of use with RNA extraction applications where we would want to keep everything cold.
We offer TRIzol reagent that will allow isolation of DNA and RNA from the same sample. Alternatively, we have the following method that has been validated by our R&D team; for sequential isolation of gDNA and total RNA from the same sample. This method involves using 2 of our kits: 1) PureLink RNA Mini Kit (Cat. No. 12183018A, 12183020, 12183025) and 2) PureLink Genomic DNA Mini Kit (Cat. No. K182002, K182000, K182001).
The protocol is detailed below:
Before starting:
- Label all spin columns and buffers from each kit with kit names to prevent confusion.
- Prepare lysis buffer and wash buffers according to the protocol from each kit.
1. Preparing lysates:
- Add 300 µL of lysis buffer (from Purelink RNA Mini Kit, beta-mercaptoethanol added) to cell or tissue sample, lyse the cells as recommended.
2. DNA isolation:
- Load all of the lysate directly onto a Purelink gDNA column (from PureLink Genomic DNA Mini Kit), save flow-through for RNA isolation.
- Centrifuge the Purelink gDNA column at 10,000 x g for 1 min.
- Wash the Purelink gDNA column with 500 µL of Wash Buffer 1 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at 10,000 x g for 1 min.
- Add 500 µL of Wash Buffer 2 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at maximum speed for 3 min to dry the membrane.
- Add 100 µL of Elution Buffer (from PureLink Genomic DNA Mini Kit), incubate at room temperature for 1 min and centrifuge at 10,000 x g for 1 min (yield can be increased if an optional second elution step is added).
- This is purified gDNA.
3. RNA isolation:
- To the above saved flow-through, add same volume of 70% ethanol, mix well and load the lysate/ethanol mix (including all precipitates) onto an RNA spin cartridge (from Purelink RNA Mini Kit).
- Centrifuge at 12,000 x g for 15 sec. Discard flow-through.
- Wash the RNA spin cartridge with 700 µL of Wash Buffer 1 (from Purelink RNA Mini Kit, ethanol added), centrifuge at 12,000 x g for 15 sec.
- Wash twice with 500 µL of Wash Buffer 2 (from Purelink RNA Mini Kit, ethanol added). After the second wash, centrifuge at 12,000 x g for 1 min to dry the membrane.
- Add 50 µL of RNase-free water onto the RNA spin cartridge, incubate at room temperature for 1 min and centrifuge at 12,000 x g for 2 min (yield can be increased if an optional second elution step is added).
- This is purified RNA.
All our TRIzol-based RNA prep kits, including the Phasemaker Tubes (Cat. No. A33248) include a chloroform-based phase separation step that cannot be skipped.
Most likely the sample was homogenized in an insufficient volume of TRIzol Reagent. Make sure to add the appropriate amount of TRIzol Reagent for your sample type.
Alternatively, the organic phase could be incompletely removed. Do not attempt to draw off the entire aqueous layer after phase separation.
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Most likely the interphase/organic phase is pipetted up with the aqueous phase. Do not attempt to draw off the entire aqueous layer after phase separation.
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Most likely the sample was not immediately processed or frozen immediately. You should process samples immediately or freeze the samples. Store RNA samples at -60 degrees C to -70 degrees C. Store DNA and protein samples at -20 degrees C.
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A lower yield can indicate that the samples may have been incompletely homogenized or lysed. You could decrease the amount of starting material or you could cut the tissue samples into smaller pieces and ensure that the tissue is completely immersed in TRIzol Reagent. Alternatively, the pellet may have been incompletely solubilized. If this is suspected, heat the sample to 50 degrees C to 60 degrees C and pipette the sample repeatedly.
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