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

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

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

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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What is the stability of TRIzol Reagent? Product FAQ

Answer

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.

Answer Id:: E2980

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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? Product FAQ

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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What is the excepted A260/A280 absorbance ratio of total RNA isolated by TRIzol Reagent? Product FAQ

Answer

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.

Answer Id:: E7831

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I see insoluble material in my tube after homogenization of my sample for RNA extraction with TRIzol Reagent. What should I do? Product FAQ

Answer

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.

Answer Id:: E7862

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Which Thermo Fisher Scientific products can be used to purify RNA from plants? Product FAQ

Answer

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.

Answer Id:: E2981

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I'm getting a low A260/280 ratio after RNA isolation with TRIzol reagent. Why? Product FAQ

Answer

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.

Answer Id:: E6553

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

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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What are some methods you suggest to isolate high-quality, intact RNA for my cDNA synthesis reaction? Product FAQ

Answer

We recommend using our Purelink RNA Mini Kit (Cat. No. 12183025) or TRIzol Reagent (Cat. No. 15596026) to isolate your RNA. Oligo(dT) selection for mRNA is typically not necessary, although it may improve the yield of specific cDNAs.

Answer Id:: E7319

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What are possible stopping points and storage for RNA extraction when using TRIzol Reagent for RNA extraction? How should I store the RNA? Product FAQ

Answer

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.

Answer Id:: E7832

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I'm getting low yield of RNA/degraded RNA when isolating it extraction with TRIzol Reagent. What could be the causes for this? Product FAQ

Answer

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.

Answer Id:: E7863

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

Answer

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

Answer Id:: E3173

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What are possible stopping points when using TRIzol Reagent? Product FAQ

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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Can I use acetone instead of isopropanol to isolate protein using TRIzol reagent? Product FAQ

Answer

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.

Answer Id:: E6554

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