iPrep™ Trizol™ Plus RNA Kit - FAQs

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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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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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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