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View additional product information for TRIzol™ Plus RNA Purification Kit - FAQs (12183555)
41 product FAQs found
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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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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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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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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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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You will need the following pipettes for the TRIzol Plus RNA Purification Kit protocol:
- 1 mL pipette: For adding TRIzol Reagent to the sample and homogenizing cells, for transferring the sample and the aqueous phase, and for adding 70% ethanol and wash buffers
- 200 µL pipette: For adding chloroform to the sample
- 30-50 µL pipette: For resuspending the RNA pellet in RNase-free deionized water
Note: Ensure that you use RNase-free pipette tips to prevent contamination and degradation of RNA during the protocol
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