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查看更多产品信息 TRIzol™ Plus RNA Purification Kit - FAQs (12183555)
53 个常见问题解答
我们的网站（https://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）中有图表对比了上述试剂盒在纯度检测和RNA完整指数（RIN）方面的差异。
TRIzol Plus RNA纯化试剂盒结合了TRIzol试剂的裂解能力和PureLink RNA小量提取试剂盒硅胶离心柱方便的RNA提取技术。
我们推荐使用纯氯仿。不需要异戊醇（尽管也可以使用比例为49:1的氯仿：异戊醇）。也可以使用含50 ppm戊烯的氯仿。此外，BCP（1-溴-2-氯丙烷）可取代氯仿。
小体积（0.5-0.8 mL）TRIzol试剂已成功用于10^2 to 10^5个细胞。但是，如果使用小体积，我们推荐使用小离心管，从而尽可能获得较高的水相柱。液体柱越高，样品被中间相污染的可能性就越小。
以下是从少量组织（1–10 mg）或细胞（100–10,000）中分离RNA的实验方案：
1.向样品中加800 μL TRIzol试剂。用移液器反复吹打以混匀细胞。直接向TRIzol试剂中加入200 μg糖原（货号10814010）。处理组织时，首先在液氮中粉碎组织，然后加入含200 μg糖原（终浓度250ug/ml）的800 μL TRIzol试剂，随后用力涡旋振荡或强力匀浆。
2.在室温下，盖上管帽并高速涡旋10秒。确保TRIzol试剂使试管边缘湿润，使所有可能残留在管壁的样品溶解。
3.剪切基因组DNA时，将样品通过连接1 mL注射器的26-gauge针两次。使用注射器将样品转移到灭菌的1.5 mL微量离心管中。
4.向每个样品中加入160 μL氯仿（或比例为49：1的氯仿：异戊醇），涡旋振荡最多30秒。将微量离心管以最大速度离心5分钟，实现液相分离。
5.将上层水相转移到新离心管中，并加入400 μL预冷的异丙醇。将样品置于–20°C沉淀1小时至过夜。将微量离心管在室温以最大速度离心15分钟，使RNA沉淀。
6.倒出上清液。使用200 μL的70%乙醇洗涤沉淀，并再次以最大速度离心10分钟。倒出上清液，在不破坏沉淀的情况下尽量移除上清液。干燥RNA沉淀。
7.使用30–50 μL无RNase的去离子水重新溶解沉淀。如果是RNase含量较高的组织（如肾上腺、胰腺），则使用100%去离子甲酰胺重悬。确保进行涡旋振荡并使用移液器反复吹打，使沉淀完全溶解。保存于–70°C。
使用TRIzol试剂提取RNA时有若干潜在停止点和推荐保存条件：
样品匀浆步骤：匀浆后（加入氯仿前），样品可在–70°C保存至少1年。匀浆后样品在加入氯仿前，可在室温保存数小时。
样品匀浆步骤：如果样品已在TRIzol试剂中有效裂解且试剂已使核酸酶失活，RNA可在室温下安全保存3-4天。
RNA沉淀步骤：异丙醇中的RNA可在4°C保存过夜。在这种低温条件下延长保存时间不会明显影响RNA产量。不要保存在-20°C下，因为盐会沉淀；不要长期保存在室温中，因为异硫氰酸胍会损害RNA。
RNA洗涤步骤：75%乙醇中的RNA在4°C可保存1周，在-20°C可保存1年。
核酸的吸光度取决于介质的离子强度和pH。请查看以下按稀释剂进行分类的吸光值范围。
稀释剂 A260 A280 A260/A280 RNA (µg/mL)
细胞质RNA溶于蒸馏水 0.381 0.223 1.711 15.24
细胞质RNA溶于TE缓冲液 0.335 0.145 2.310 13.4
使用TRIzol试剂分离的RNA溶于蒸馏水 0.585 0.328 1.785 23.4
使用TRIzol试剂分离的RNA溶于TE缓冲液 0.544 0.247 2.206 21.76
尽管高A260/A280比值可能并不代表核酸样品非常纯，但低A260/A280比值（RNA为1.7）确实代表样品存在污染并且可能不适合某些应用。
如果水相完全去除并向样本中加入乙醇，乙醇会因密度较低而位于TRIzol试剂的上方。如果未充分混合酒精及有机相就对样本进行离心，则在离心后乙醇位于TRIzol试剂的上方，DNA仍处于中间相，而TRIzol试剂位于底部的红色有机部分。如果没有恰当混合乙醇，则应继续混合样本，然后离心并继续完成DNA分离实验方案中的步骤1。
如果不慎加入了70%乙醇，则可能在有机相上方出现小体积的水相。由于实验方案所用洗涤液含有不超过30%的水，因此，在有机相上方出现的水层不会超过0.3mL的30%（90 µL）。您可在继续分离前尝试去除水相，但是DNA得率可能会降低。
如果在RNA分离过程中出现水相与有机相分离不完全，也会出现这种情况，原因主要有四个：1，氯仿混合不充分；2，样本离心时转速不合适；3，未达到需要的离心时间，4，离心温度错误。这些原因导致最终将从样本中回收到明显少于600 µL的RNA水相。液相分离问题通常出现于使用涡旋振荡来混合试管中的氯仿时。由于TRIzol试剂和有机相之间较大的密度差异，这些溶液很难完全被混匀，因此只能回收部分水相。当加入乙醇并再次充分混合样本时，将再次出现水相和有机相的分离，并且水相会出现于样本上方。
通常，吸光度低是由苯酚污染所致。这在用室温离心代替4°C离心样品时时有发生。我们推荐采取二次乙醇沉淀去除残留苯酚。
以下是DNA得率很低或者DNA发生降解的几种可能的原因：
•加入TRIzol后，样本未完全均质或裂解。如果在加入氯仿后有任何固体样品残留，DNA得率可能很低，因为DNA依然停留在非均质的样本中。您可以使用聚丙烯滤布过滤TRIzol匀浆（在液相分离前），从而去除未完全裂解的样品残留。
•最终的DNA沉淀未能完全溶解。溶解DNA可能需要数小时。在37°C孵育同时反复吹打几次可能对DNA的溶解有所帮助。同时，应确保DNA浓度不会过浓否则可能不能完全溶解。如果DNA未完全溶解，那么在离心时去除未溶解的胶样样品时，会损失一些DNA。
•动物或其他来源的组织，在获取后未立即处理或冻存。
•使用高速匀浆机匀浆样本。可能发生DNA断裂。
•如果预期得率<10 μg，可能是沉淀过程的物理操作本身的限制导致的。匀浆和/或清洗步骤可使用微载体（糖原、tRNA），或将样本合并以提高预期得率。
DNA难以溶解最常见原因是DNA沉淀被干燥过度。DNA沉淀干燥一定不要超过10分钟。如果您遵从以下简单建议，则可避免许多核酸溶解的问题。
使用灭菌棉球除去试管壁上的乙醇液滴。其他乙醇可通过使用灭菌毛细移液管尖接触DNA沉淀而去除。多余的乙醇将被毛细作用吸到移液管内。DNA沉淀中残留的乙醇不会产生负作用。通常，在DNA沉淀完全干燥前加入TE缓冲液或8 mM NaOH，可解决DNA溶解问题。DNA沉淀会在孵育5-10分钟后溶解而变透明。为了完全溶解DNA，可在定量前使用移液器反复吹打DNA溶液。 如需将干燥过度的DNA团块溶解，可以将其置于冰箱，并定期用移液器吹打至DNA沉淀变为透明并完全溶解于溶液中。 多糖为水溶性的，可随RNA进入水相。另外，在非过度干燥的情况下，受到污染的RNA和DNA沉淀比非常纯的沉淀更易溶解。在8 mM NaOH中不溶解的团块，也不会溶解于苯酚/氯仿溶液。可以，单链DNA将在DNA相中分离。
苯酚相和中间相可于4°C条件下过夜储存。样本也可在清洗液（用10%的酒精配置的0.1 M柠檬酸钠）中储存至少几个小时。也可将样本重悬于75%的乙醇中，在4°C条件下储存数月。
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within ourRNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
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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