TRIzol™ Reagent, 200 Preps - FAQs

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我在使用TRIzol试剂提取RNA后，得到了较高的A260/A280比值，有哪些原因？

降解的RNA可引起260 nm处的吸光度升高。

加入氯仿后（加入异丙醇前），离心后在试管底部出现沉淀，可能有哪些原因？

沉淀最可能是多糖或细胞膜；DNA应该在中间相。在含血液的样品（如，肝脏）中，可能在沉淀顶部出现可见的红色粘稠层。这最可能是血液产物，不应与上清液一起转移。

使用TRIzol试剂进行相分离时，出现黄棕色或浅粉色的水相，可能有哪些原因？

•这在皮肤样品中很常见。据推测，皮肤样品中含有脂肪，脂肪微粒在离心时会漂浮。在皮肤样品中，微粒会粘附黑色素，使水相产生颜色。脂肪微粒也可从TRIzol试剂中粘附色素，产生浅粉色。如果认为样品中含有脂肪，则在加入氯仿前对TRIzol试剂处理的匀浆液进行离心。脂肪会在上清液顶层形成透明层；应使用移液器将这一层移走并丢弃。
•若样品含有很多血液，则水相可能会变浑浊和/或淡黄色（可能由于血红蛋白含有铁）。如果离心机不是低温，有机相会变成深栗色；其中一些颜色会进入水相，使水相变为橙色或黄色。
•出现浅粉色水相的原因也可能是样品过度稀释（即，样品与TRIzol试剂的比例> 1:10）或者样品中含有过多的盐和蛋白质。这会引起过早的相分离，可通过在样品中多加入一点TRIzol试剂进行补救。从浅粉色水相中分离的RNA，很可能存在DNA污染。

我从FFPE组织中分离RNA时，RNA质量和得率非常差。该如何提高RNA总体质量和得率？

以下是我们的建议：

1.上游组织获取和组织样品制备——尽可能在手术切除后1小时内固定组织。完成固定步骤前，可能会发生大量RNA降解。最佳固定时长为12-24小时，应使用中性福尔马林或多聚甲醛溶液。在包埋前，固定组织应完全脱水。
2.整块储存——尽可能整块保存，无切割面，以防止暴露于大气中的氧气、水以及光、感染（真菌、昆虫等）等其他环境因素造成持续伤害。
3.RNA分离时组织类型、大小和用量的选择——推荐的组织厚度为10–20 µm。切片数取决于组织类型（影响细胞密度）和表面积（推荐大小：50–300 mm^2）。过多的起始材料可引起滤膜堵塞，产生较差的得率。
4.避免使用过量的石蜡进行组织包埋——尽可能在开始纯化步骤前去除过多的石蜡。对于二甲苯纯化法，室温下2次二甲苯处理应足以完全脱蜡。如果需要，可采取更严格的处理，37–55°C最多孵育30分钟。在二甲苯脱蜡后，一定要完全移去100%乙醇，并在2次100%乙醇洗涤后干燥沉淀。磁珠法利用新型化学反应处理石蜡，将输入量限制到20 µm的切片。

点击 此处（https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-sample-extraction/working-with-ffpe-samples.html），阅读关于从FFPE组织中分离RNA的更多信息。

我的样品具有高含量的蛋白多糖和/或多糖。需要对RNA分离的TRIzol实验方案进行任何改进吗？

若已知样品具有高含量的蛋白多糖和/或多糖（如大鼠肝脏、大鼠主动脉、植物），对RNA沉淀步骤做如下改进应该可以从分离的RNA中除去上述污染物：

向每1 mL TRIzol试剂匀浆得到的水相中加入0.25 mL异丙醇，随后加入0.25mL高盐沉淀液（0.8M柠檬酸钠和1.2 M NaCl；无需调整pH）。将溶液混合、离心，并继续使用实验方案中的方法分离。
这种改进的沉淀方法可有效沉淀RNA并维持蛋白多糖和多糖的溶解性。为了从多糖含量很高的植物材料中分离纯化RNA，在使用改进沉淀方法的同时，应对初始匀浆液进行额外的离心。通常，在多糖或蛋白多糖污染不会带来问题时，我们不推荐使用高盐沉淀法，因为这个额外步骤并不会带来任何显著优势。一般来说，在多糖或蛋白多糖污染不会带来问题的情况下纯化RNA样品时，使用或不使用高盐沉淀液，总RNA得率都一样。RNA谱会有细微变化，体现在tRNA量的轻微降低。高盐沉淀会使样品中的tRNA减少。

在TRIzol分离程序后，未出现RNA沉淀。你们有什么诀窍吗？

在样品中加入糖原有助于改善得率并能和RNA一起沉淀下来（糖原是水溶性的）。聚丙烯酰胺也可用作沉淀少量RNA的载体。另外也可使用鲑鱼精DNA，在沉淀水相时加入。

我使用TRIzol试剂进行提取后发现RNA样品的A260/A280比值很差(<1.65)。可能的原因有哪些？

•样品在过少的TRIzol试剂中匀浆。
•匀浆后，未将样品在室温下保存5分钟。（这可能导致核蛋白未解离）。
•最终得到的RNA沉淀未完全溶解。这可能是因为RNA沉淀干燥过度（如果沉淀是透明的而非白色，则表示干燥过度）。为了使沉淀完全溶解，应加热至55–60°C维持10–15分钟，并反复吹打。
•可能存在苯酚污染。这可能是因为样品是在室温下离心，而非4°C；苯酚在室温下更易溶于水相。如果在270 nm有吸光度（由于存在苯酚），则可通过乙醇沉淀除去样品中的残留苯酚。
•胍类约在240 nm处有吸光值。苯酚有2个吸收峰：一个约为275nm，另一个宽峰范围为220 nm以下至240nm左右。如果在此范围内观察到很大的峰，则建议再次沉淀和洗涤。为防止发生这种情况，我们还是建议加入氯仿后在4°C的进行相分离。
•可能有氯仿残留；应再次沉淀。
•在一些溶于水的样品中，水的酸性或低离子含量可能导致A260/A280比值较低。若样品溶于TE中，并使用TE缓冲液（或1–3 mM Na2HPO4，pH ~8.0）调零分光光度计，则该比率会升高。核酸的摩尔消光系数是在中性pH条件下得到的，这表明在中性pH条件下260nm处的吸光度最高。
•不同的分光光度计会带来A260/A280比值差异（可能原因是，在260 nm“调零”后，仪器在280 nm的“调零”方法会产生不同的A280值）。在这种情况下，我们建议使用不同的分光光度计。

使用TRIzol试剂提取后，在RNA制品中看到DNA污染。可能原因有哪些？

在最初的分离步骤后，一部分中间相随水相一起被移出了。出现这种情况的原因有多种：

- 加入到样品中的TRIzol试剂量不足。通常，每0.05 g组织或每10 cm2培养皿应使用1 mL TRIzol试剂。
- 原始样品中可能含有痕量的其他有机物质（乙醇、DMSO等）。
- 在RT-PCR前，应使用扩增级纯度的DNase I处理RNA。
- 在首次匀浆后、氯仿提取前，可能有未被离心除去的不溶物。
- 从转染了质粒的细胞中分离的RNA中可能含有DNA污染，则有可能是质粒的污染，因为在向TRIzol试剂中加入氯仿后，不是所有质粒DNA都可能进入到中间相/有机相。若使用RT-PCR检测转染质粒的细胞中基因的表达，则需要进行DNase I处理。

使用TRIzol试剂分离提取时，RNA得率较低或 RNA出现降解，可能是什么原因造成的？

请查看以下RNA得率较低或得到降解的RNA的原因：

•在最后的乙醇沉淀步骤中，可能使用过SpeedVac系统浓缩RNA或冻干过RNA。完全干燥的RNA溶解性降低。此外，如果使用了过高的离心速度（高于12,000 x g），也会导致RNA/DNA难溶解。
•RNA沉淀可能未完全溶解。为提高溶解率，使用移液器将沉淀在SDS溶液或DEPC水中反复吹打，然后加热至50–60°C。样品可能富含多糖或蛋白多糖。如果是这样，则异丙醇沉淀步骤应使用0.25体积异丙醇和0.25体积高盐溶液。
•加入TRIzol试剂前，洗涤了细胞。在加入TRIzol试剂前洗涤细胞，会增加mRNA降解的几率。
•样品未完全匀浆。
•从动物或其他来源取出组织后，未立即分离RNA或冻存。
•组织未完全裂解；如果在加入氯仿前离心，会出现白色粘液状沉淀。如果出现棕褐色沉淀，则表示细胞未完全裂解。
•如果使用研钵和研杵研磨组织，则RNA和DNA可能非特异性粘附在研钵和研杵上。最好使用玻璃匀浆仪和Teflon研杵；向匀浆仪中加入TRIzol试剂，然后加入冻存的组织并开始匀浆。
•分离后的RNA可能被储存在–20°C，而不是–70°C下。
•曾使用胰酶裂解组织培养细胞。
•连续不断长时间在小体积样品（如，1 mL）中匀浆可能引起样品升温；这会导致组织中RNA降解。匀浆时应冷却样品，并采取间断性匀浆（与连续匀浆相反）。
•定量前，样品的保存液和稀释液不同，会使OD读取结果不同。这也会导致得率低的假象。
•过多的RNAlater溶液（>0.05 mL）会降低RNA回收率，导致相分离出现问题。

使用TRIzol试剂提取RNA时，样品匀浆后管中有不溶性物质，该如何处理？

有2种方法可以去除不溶性物质：

1.仅分离RNA：如果经过匀浆并在室温静置5分钟后有很多不溶性物质，则可在加入氯仿前通过4°C12,000 x g离心10分钟去除不溶物（应出现透明的上清液和果冻样沉淀）。将上清液转移至新管子，并继续进行下一步。注意：若后续计划进行DNA分离，则不应这样做。
2.RNA和DNA分离：如果经过匀浆并在室温静置5分钟后有很多不溶性物质，则可将匀浆物通过聚丙烯滤膜以去除可能影响DNA沉淀的不溶性物质。

是否有任何资料显示使用TRIzol试剂、PureLink RNA小量提取试剂盒、ChargeSwitch总RNA细胞试剂盒或TRIzol Plus RNA纯化试剂盒提取RNA存在区别？

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纯化试剂盒和TRIzol试剂有何区别？

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。

我想从同一样品中分离RNA和蛋白质，你们有这样的试剂盒吗？

有，您可使用TRIzol试剂或mirVana PARIS RNA和非变性蛋白质纯化试剂盒。

使用TRIzol试剂提取时，在加入100%乙醇后会出现水相。为什么会出现这种情况？

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。

在使用TRIzol试剂提取后，发现DNA的 (A260/A280)比值很低，<1.70。原因是什么？

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。

在使用TRIzol试剂分离DNA时发现DNA得率很低或者DNA发生了降解，这是为什么？

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。

我在使用TRIzol试剂时，发现提取的DNA难以溶解，该如何处理？

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。

能使用TRIzol试剂分离单链DNA么？

可以，单链DNA将在DNA相中分离。

应用TRIzol试剂时，哪几个时间点可暂停操作？

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。

I'm getting a high A260/A280 ratio for RNA after extraction with TRIzol Reagent. What could be the cause of this?

Degraded RNA can cause an increased absorbance at 260 nm.

Find additional tips, troubleshooting help, and resources within ourRNA Sample Collection, Protection, and Isolation Support Center.

I'm getting a precipitate at the bottom of the tube following centrifugation after adding chloroform (before isopropanol was added). What could be the cause of this?

This is most likely polysaccharides or cell membranes; DNA should be in the interphase. In samples containing blood (e.g., liver), a red viscous layer may be visible on top of the pellet. This is most likely due to blood products and should not be carried over with the supernatant.

During phase separation with TRIzol Reagent, I see a yellowish-brown or pinkish aqueous phase. What is causing this?

- This is common with skin samples. It is assumed that there is fat in these samples, and the fat micelles float during the centrifugation. 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 and cause 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.
- 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). 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.
- A pinkish aqueous phase may also be caused by overdilution of the sample (i.e., a sample to TRIzol Reagent ratio > 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.

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

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).

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?

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.

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

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

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

- 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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I'm seeing DNA contamination in my RNA prep after extraction with TRIzol Reagent. What could be the cause of this?

A portion of the interphase may have been removed with the aqueous phase after the initial separation. This can occur for several reasons.

- An insufficient amount of TRIzol Reagent was added to the sample. In general, 1 mL of TRIzol Reagent should be used for every 0.05 g of tissue or every 10 cm2 dish.
- The original sample may have had traces of other organic material to begin with (ethanol, DMSO, etc.).
- RNA should be treated with amplification grade DNase I prior to RT-PCR.
- There may have been insolubles after the first homogenization that were not removed by centrifugation before chloroform extraction.
- If the DNA contamination was detected in RNA isolated from cells after transfection with a plasmid, not all of the plasmid DNA may have partitioned into the interphase/organic phase once the chloroform was added to the TRIzol Reagent. If RT-PCR is being used to assay gene expression from the transfected plasmid, a DNase I treatment will be needed.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I'm getting low yield of RNA/degraded RNA when using TRIzol Reagent. What could be the causes for this?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

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

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.

Do you have any data showing differences in RNA extraction with TRIzol Reagent, the PureLink RNA Mini Kit, ChargeSwitch Total RNA Cell Kit or the TRIzol Plus RNA Purification Kit?

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.

What is the difference between the TRIzol Plus RNA Purification Kit and TRIzol Reagent?

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.

I'd like to isolate RNA and protein from the same sample. Do you have a kit for this?

Yes, you can use TRIzol Reagent or the mirVana PARIS RNA and Native Protein Purification Kit.

I'm getting an aqueous layer that appears after addition of 100% ethanol during extraction with TRIzol Reagent. Why is this happening?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I'm getting low absorbance for DNA (A260/A280) ratio <1.70 after TRIzol Reagent extraction. What could be causing this?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I'm getting low yield of DNA/DNA degradation when isolating it with TRIzol Reagent. What happened?

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.

I'm having problems with my DNA solubilizing into solution when using TRIzol Reagent. What can I do?

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.

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

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

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What are possible stopping points when using TRIzol Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What is the difference between TRIzol reagent and TRIzol Plus reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

Can I precipitate small RNA species using TRIzol reagent?

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.

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

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What do you recommend for long-term storage of RNA?

For long-term storage, we recommend resuspending the RNA in stabilized formamide and storing at -70 degrees C. To remove the formamide, add 4 volumes ethanol and, if less than 20 µg RNA, also add NaCl to 0.2 M. Precipitate the RNA and use for downstream experiments.

Can I use a DNA carrier with TRIzol reagent? If so, when can I add it to the protocol?

Yes, you can use salmon sperm DNA as a carrier. Add it during the precipitation of the aqueous phase.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

The TRIzol Reagent protocol specifies centrifugation speeds of 12,000 x g for 10 mins for RNA precipitation, but my centrifuge only goes up to 5,000 x g. Can I still perform my experiment?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I accidentally added too much chloroform to my TRIzol Reagent reaction. What should I do?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

I inadvertently added isopropanol instead of chloroform. What should I do?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What carrier do you recommend for isolation of RNA with TRIzol Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

At what points can I stop in the protocol for TRIzol Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

Do you have recommendations for homogenizing my sample for RNA isolation with TRIzol Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

Can I use TRIzol Reagent to isolate RNA from tissue that was stored in RNAlater Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What is the difference between your TRIzol Reagent and TRIzol LS Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What does the TRI stand for in TRIzol Reagent?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What is the composition of your TRIzol Reagent?

TRIzol Reagent is a ready-to-use mixture of phenol, guanidine isothiocyanate, red dye, and other proprietary components.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

Can I use TRIzol Reagent to isolate RNA from a sucrose gradient fraction?

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).

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

How does TRIzol Reagent work for RNA isolation?

TRIzol Reagent contains phenol and guanidine isothiocyanate, which allow for isolation of nucleic acids from proteins. Further partitioning of the nucleic acids occurs in a pH dependent manner. At pH 7.0 or higher, DNA and RNA partition into the aqueous phase. At an acidic pH, below 7.0, DNA is denatured and will move into the organic phase (interphase) yet the RNA remains in the aqueous phase.TRIzol Reagent has pH about 5.

If blood has been collected in EDTA, is the efficiency of the TRIzol Reagent RNA isolation procedure affected?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

After I add chloroform to the TRIzol Reagent mixture, the aqueous phase is pinkish in color. What is the reason behind this pinkish coloration and will it effect the quality of my RNA?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What is the difference between TRIzol and TRIzol LS Reagent?

TRIzol LS Reagent is a complete, ready-to use reagent for easy and simultaneous isolation of total RNA, DNA, and proteins from liquid samples. The reagent, a mono-phasic solution of phenol and guanidinium thiocyanate, is an improvement to the single-step RNA isolation method developed by Chomzcynski and Sacchi. TRIzol LS Reagent is similar to the original TRIzol Reagent in composition and results of use. TRIzol LS Reagent is designed for use with liquid samples such as blood and virus preparations in which large volumes of aqueous samples need to be processed, while TRIzol Reagent is designed for cell cultures or tissues. It is formulated to accommodate processing of more liquid sample per unit of reagent compared to the original formula.

The only difference between TRIzol Reagent and TRIzol LS Reagent is the concentration of components. TRIzol  LS Reagent is slightly more concentrated. The formula allows lower quantities of reagent to be used relative to a liquid sample. (TRIzol = 10:1 required, TRIzol LS = 3:1 required). If you buy TRIzol LS Reagent, but want to use it like TRIzol Reagent (on solid samples), there will probably be a decrease in yield vs. using regular TRIzol Reagent. TRIzol LS Reagent should NOT be used undiluted with solid samples. To dilute: take 750 µL TRIzol LS Reagent + (50 to 100 mg tissue + water to make 250 µL).

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

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

I want to extract DNA, and if possible, RNA from formalin-fixed specimens in paraffin blocks. Which product would work for me?

We recommend the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975). This kit is optimized for isolation of both DNA and RNA from formalin or paraformalin-fixed, paraffin-embedded (FFPE).

Another option is TRIzol Reagent, but be sure to check the references listed below. Because paraffin is not soluble in TRIzol Reagent, paraffin-embedded tissues can be quick-heated to get the tissue out of the paraffin; any paraffin which remains will float to the top of the aqueous phase (and should be avoided). (If the slice is very thin, the whole slice can be added to the TRIzol Reagent, and hopefully, the tissue will be exposed to the reagent). Most of the references we surveyed do not provide quantitative data, because paraffin-embedded tissues are dramatically influenced by the action of nucleases prior to fixation and by the formalin fixation time.

The ability to detect specific housekeeping genes by PCR analysis with RNA or DNA extracted from these tissues is usually considered to be a positive result. We do not have a protocol per se, but we have spoken with customers who are doing this. We recommend deparaffinizing with xylene (or other organic), then grinding the sample very thoroughly in TRIzol Reagent (may require a Polytron); in most cases, you have to homogenize with vigor because the DNA is crosslinked and you have to get it free. Microcarrier is recommended since the RNA is crosslinked and fragmented. From this point, the standard isolation protocol can be used. They have found publications that show that the success of the isolation is dependent on how long the sample was fixed (there is an inverse relationship): Inoue, T., et. al., Pathology International (1996) Vol 46, Iss 12, pp. 997-1004.

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

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.)

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

What can I do to increase the RNA yield using TRIzol Reagent from small amounts of starting samples?

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.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

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?

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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Can TRIzol Reagent be used to isolate RNA from small samples?

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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Can purifications with TRIzol Reagent be scaled up?

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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How can I increase the stability of the RNA isolated from TRIzol Reagent during storage?

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

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

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Can I use a table-top centrifuge when I use the TRIzol Reagent?

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

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

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

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.

Can DNA purified with TRIzol Reagent be used in restriction digests or PCR reactions?

Yes. However, since the DNA is dissolved in 8 mM NaOH, the pH needs to be adjusted with HEPES. Please contact Technical Service for details. Depending on the restriction endonuclease, use 3 to 6 units per µg of DNA for between 3 hr and overnight. In a typical assay, 80 to 90% of the DNA is digestible.

Can I add an antifoaming agent to TRIzol Reagent to prevent foaming when grinding tissues with a homogenizer?

There are already components of the TRIzol Reagent that function as anti-foaming agents. The addition of other anti-foaming agents has not been tested. If you attempt adding other components, you may experience higher DNA contamination. If you are working with very small samples, we might suggest using a slightly larger volume of TRIzol Reagent in order to minimize loss of sample when homogenizing. Of course, as always, use caution when adding anything to an RNA purification reagent as RNA degradation is always a concern.

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

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.

What is the stability of TRIzol Reagent?

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.

Will the stability of TRIzol Reagent be affected if it was left at room temperature overnight?

No, the stability of TRIzol Reagent will not be impacted by storing it at room temperature overnight. The 4 degrees C storage condition for the reagent helps with ease of use with RNA extraction applications where we would want to keep everything cold.

Do you offer a kit that will allow me to sequentially isolate gDNA and total RNA from my tissue sample that is not an FFPE (formalin-fixed, paraffin-embedded) sample?

We offer TRIzol reagent that will allow isolation of DNA and RNA from the same sample. Alternatively, we have the following method that has been validated by our R&D team; for sequential isolation of gDNA and total RNA from the same sample. This method involves using 2 of our kits: 1) PureLink RNA Mini Kit (Cat. No. 12183018A, 12183020, 12183025) and 2) PureLink Genomic DNA Mini Kit (Cat. No. K182002, K182000, K182001).

The protocol is detailed below:

Before starting:
- Label all spin columns and buffers from each kit with kit names to prevent confusion.
- Prepare lysis buffer and wash buffers according to the protocol from each kit.
1. Preparing lysates:
- Add 300 µL of lysis buffer (from Purelink RNA Mini Kit, beta-mercaptoethanol added) to cell or tissue sample, lyse the cells as recommended.

2. DNA isolation:
- Load all of the lysate directly onto a Purelink gDNA column (from PureLink Genomic DNA Mini Kit), save flow-through for RNA isolation.
- Centrifuge the Purelink gDNA column at 10,000 x g for 1 min.
- Wash the Purelink gDNA column with 500 µL of Wash Buffer 1 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at 10,000 x g for 1 min.
- Add 500 µL of Wash Buffer 2 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at maximum speed for 3 min to dry the membrane.
- Add 100 µL of Elution Buffer (from PureLink Genomic DNA Mini Kit), incubate at room temperature for 1 min and centrifuge at 10,000 x g for 1 min (yield can be increased if an optional second elution step is added).
- This is purified gDNA.

3. RNA isolation:
- To the above saved flow-through, add same volume of 70% ethanol, mix well and load the lysate/ethanol mix (including all precipitates) onto an RNA spin cartridge (from Purelink RNA Mini Kit).
- Centrifuge at 12,000 x g for 15 sec. Discard flow-through.
- Wash the RNA spin cartridge with 700 µL of Wash Buffer 1 (from Purelink RNA Mini Kit, ethanol added), centrifuge at 12,000 x g for 15 sec.
- Wash twice with 500 µL of Wash Buffer 2 (from Purelink RNA Mini Kit, ethanol added). After the second wash, centrifuge at 12,000 x g for 1 min to dry the membrane.
- Add 50 µL of RNase-free water onto the RNA spin cartridge, incubate at room temperature for 1 min and centrifuge at 12,000 x g for 2 min (yield can be increased if an optional second elution step is added).
- This is purified RNA.

Can I skip the phase separation step in your TRIzol-based RNA prep kits?

All our TRIzol-based RNA prep kits, including the Phasemaker Tubes (Cat. No. A33248) include a chloroform-based phase separation step that cannot be skipped.

Why is the A260/280 ratio of my sample, extracted using the TRIzol Reagent, low?

Most likely the sample was homogenized in an insufficient volume of TRIzol Reagent. Make sure to add the appropriate amount of TRIzol Reagent for your sample type.
Alternatively, the organic phase could be incompletely removed. Do not attempt to draw off the entire aqueous layer after phase separation.

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Why is my RNA, extracted using the TRIzol Reagent, contaminated?

Most likely the interphase/organic phase is pipetted up with the aqueous phase. Do not attempt to draw off the entire aqueous layer after phase separation.

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Why is my sample, extracted using the TRIzol Reagent, degraded?

Most likely the sample was not immediately processed or frozen immediately. You should process samples immediately or freeze the samples. Store RNA samples at -60 degrees C to -70 degrees C. Store DNA and protein samples at -20 degrees C.

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Why is my yield lower than expected from samples extracted using the TRIzol Reagent?

A lower yield can indicate that the samples may have been incompletely homogenized or lysed. You could decrease the amount of starting material or you could cut the tissue samples into smaller pieces and ensure that the tissue is completely immersed in TRIzol Reagent. Alternatively, the pellet may have been incompletely solubilized. If this is suspected, heat the sample to 50 degrees C to 60 degrees C and pipette the sample repeatedly.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.