TRIzol™ LS Reagent, 100 mL - FAQs

We recommend using straight chloroform. No isoamyl alcohol is needed (though using chloroform:isoamyl alcohol 49:1 works without problems). You can also use chloroform with 50 ppm amylene. Alternatively, BCP (1-bromo-2 chloropropane) can be used in the place of chloroform.

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Small volumes (0.5-0.8 mL) of TRIzol Reagent have been used successfully for 10^2 to 10^5 cells, but if small volumes are used, we recommend using smaller tubes in order to have the tallest possible column of aqueous phase. The taller the column of liquid, the less likely that contamination from the interphase will occur.

Here is a protocol for isolation of RNA from small quantities of tissue (1-10 mg) or cells (100-10,000):
1. Add 800 µL TRIzol Reagent to the sample. Homogenize cells by pipetting repeatedly. Add 200 µg glycogen (Cat. No. 10814010) directly to the TRIzol Reagent. If processing tissue, pulverize in liquid nitrogen first and then add 800 µL TRIzol Reagent containing 200 µg glycogen (final concentration 250 µg/mL) followed by vigorous vortexing or power homogenization.
2. Place at room temperature, cap the vial, and vortex at high speed for 10 seconds. Make sure the TRIzol Reagent wets the side of the vial in order to solubilize any sample that may be remaining on the walls.
3. Shear the genomic DNA in the sample by passing twice through a 26-gauge needle connected to a 1 mL syringe. Using the syringe, transfer the sample to a sterile 1.5 mL microcentrifuge tube.
4. Add 160 µL of chloroform (or 49:1 chloroform:isoamyl alcohol) to each sample and vortex up to 30 seconds. Centrifuge at maximum speed in a microcentrifuge for 5 minutes to separate the phases.
5. Transfer the upper aqueous phase to a fresh tube and add 400 µL ice-cold isopropanol. Allow the samples to precipitate at -20 degrees C for 1 hour to overnight. Pellet the RNA by centrifugation at maximum speed in the microfuge for 15 minutes at room temperature.
6. Decant the supernatant. Wash the pellet in 200 µL of 70% ethanol and centrifuge again for 10 minutes at maximum speed. Decant the supernatant, removing as much as possible without disturbing the pellet. Dry the RNA pellet.
7. Resolubilize the pellet in 30-50 µL RNAse-free deionized water. If tissue is high in RNAses (e.g., adrenal gland, pancreas), resuspend in 100% deionized formamide. Be sure to vortex or pipette the sample up and down to ensure that the pellet is fully resolubilized. Store at -70 degrees C.

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There are several possible stopping points and recommended storage conditions during the extraction of RNA with TRIzol Reagent:

- Sample homogenization step: After homogenization (before addition of chloroform), you can store samples at -70 degrees C for at least 1 year. The homogenated samples can sit at room temperature for several hours before adding chloroform.
- Sample homogenization step: If samples are efficiently lysed in TRIzol Reagent and the reagent inactivates the nucleases, you can safely store RNA for 3-4 days at room temperature.
- RNA precipitation step: You can store RNA in isopropanol overnight at 4 degrees C. Prolonged storage at this reduced temperature will not influence the yield of RNA appreciably. Do not store at -20 degrees C, as salts will precipitate, and do not store for a prolonged time at room temperature because the guanidine isothiocyanate can harm the RNA.
- RNA wash step: You can store RNA in 75% ethanol for 1 week at 4 degrees C or 1 year at -20 degrees C.

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The absorbance of nucleic acids is dependent upon the ionic strength and pH of the medium. Please see the range of absorbance values below based on the diluents used.

Diluent A260 A280 A260/A280 RNA (µg/mL)
Cytoplasmic RNA dissolved in distilled water 0.381 0.223 1.711 15.24
Cytoplasmic RNA dissolved in TE buffer 0.335 0.145 2.310 13.4
RNA isolated by TRIzol Reagent and dissolved in distilled water 0.585 0.328 1.785 23.4 RNA isolated by TRIzol Reagent and dissolved in TE buffer 0.544 0.247 2.206 21.76 Although a high A260/A280 ratio may not indicate an extremely pure preparation of nucleic acid, a low A260/A280 ratio (1.7 for RNA) does indicate that the preparation is contaminated and may not be suitable for some applications.

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Please visit our website for tips for working with blood samples.

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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.

Yes, as long as the antibody used binds to the target protein in its denatured form.

The reagent to sample ratios for TRIzol Reagent (Cat. No. 15596026) and TRIzol LS Reagent (Cat. No. 10296010) are as follows:

• For TRIzol LS Reagent - 3:1 (0.75 mL of TRIzol LS Reagent with 0.25 mL of sample)
• For TRIzol Reagent- 10:1 (sample volume should not exceed 10% of the volume of TRIzol Reagent)

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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. 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.

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 (e.g., TRIzol = 10:1 required, TRIzol LS = 3:1 required). If you want to use TRIzol LS Reagent with solid samples, there will probably be a decrease in yield compared to using the 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).

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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.