Regulatory requirements vary by product. Please see product-specific literature to determine product use.

Product FAQ

What are possible stopping points when using TRIzol Reagent?

Answer

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.

Answer Id: E7675

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Product FAQ

Can I use TRIzol Reagent to isolate single-stranded DNA?

Answer

Yes, single-stranded DNA will separate with the DNA phase.

Answer Id: E7676

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Product FAQ

Can proteins that are purified with TRIzol Reagent be immunoprecipitated?

Answer

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

Answer Id: E2966

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Product FAQ

I am getting a poor A260/A280 ratio for RNA (<1.65) after extraction with TRIzol Reagent. What could be the cause of this?

Answer

- 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.

Answer Id: E7865

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Product FAQ

I don’t see an RNA pellet after the TRIzol isolation procedure? Do you have any tips?

Answer

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.

Answer Id: E7866

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Product FAQ

My sample has a high content of proteoglycans and/or polysaccharides. What are your suggestions when performing TRIzol extraction?

Answer

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.

Answer Id: E6544

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Product FAQ

My sample has a high content of proteoglycans and/or polysaccharides. Are there any modifications that I should make to the TRIzol protocol for RNA isolation?

Answer

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.

Answer Id: E7867

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Product FAQ

Why do I lose my DNA pellet after washing with 70% ethanol?

Answer

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.

Answer Id: E3113

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Product FAQ

Can I precipitate small RNA species using TRIzol reagent?

Answer

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.

Answer Id: E6545

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Product FAQ

I isolated RNA from FFPE tissue and got very poor RNA quality and yield. How can I improve the overall RNA quality and yield?

Answer

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).

Answer Id: E7868

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Product FAQ

Do you have a reagent that will isolate RNA, DNA, and protein from the same sample?

Answer

Our TRIzol Reagent (Cat. No. 15596026) can isolate RNA, DNA, and protein from the same sample.

Answer Id: E3226

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Product FAQ

How can I avoid genomic DNA contamination when isolating RNA with TRIzol Reagent?

Answer

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.)

Answer Id: E3191

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Product FAQ

What type of tubes should I use with the TRIzol Reagent?

Answer

Use polypropylene tubes. Do not use tubes sensitive to phenol.

Answer Id: E3176

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Product FAQ

If my tissue has a high content of proteoglycans and/or polysaccharides, what can I do to ensure that these compounds don't contaminate the RNA I get from my TRIzol Reagent purification?

Answer

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.

Answer Id: E3189

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Product FAQ

What will be the approximate aqueous volume when using TRIzol Reagent?

Answer

About 60% of the TRIzol Reagent volume becomes part of the aqueous phase after chloroform addition.

Answer Id: E3173

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