Lipofectamine™ MessengerMAX™ Transfection Reagent, 1.5 mL - FAQs

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我正在尝试解决用Lipofectamine Messenger MAX转染试剂转染mRNA操作中遇到的疑难。你们能告诉我一些转染后未见表达的可能原因吗？我们使用的是原代细胞，所以我不确定这些细胞是否自身就很难被转染，还是另有原因。

转染后有一些因素会影响表达，但在逐一排除这些可能性之前，应使用GFP mRNA阳性对照(Tri-Link CleanCap EGFP mRNA, 货号L-7201)和Lipofectamine MessengerMAX mRNA转染试剂来尝试转染实验。 如果这一操作都未获得良好的转染结果，最好尝试使用一种替代的导入方法或换用另一种细胞。不过，如果这一实验获得了可接受的结果，下一步则可尝试使用感兴趣的mRNA和MessengerMAX试剂进行转染。如果此时仍未获得理想的表达水平，则可能是所使用mRNA的问题，故障排除可能需要从此方面出发来考虑。举例来说，一些可能涉及的问题包括：有5’帽吗？有多聚A尾吗？mRNA纯度如何？是否在凝胶电泳中获得了单一条带？DNA模板是否纯净？

我们观察到转染效率随着细胞传代次数不同而发生很大变化。你们有推荐的最佳传代次数吗？

在不同代次间看到转染效率的一些差异很正常。为了将这一差异降到最低，我们推荐使用5-20代的细胞进行转染实验。在每一个转染实验中都包括阳性GFP mRNA对照(Tri-Link CleanCap EGFP mRNA, 货号L-7201)有助于定量确定和跟踪每周转染效率的变化。

我在mRNA转染的过程中发现了某些细胞毒性的存在。你们能就此提供一些降低毒性的建议吗？

考虑在mRNA模板中包括适当的5’UTR和3 & # 146;UTR序列以加强mRNA的稳定性和细胞的接受度。使用MegaClear转录纯化试剂盒纯化的高纯度的体外转录mRNA。确保A260/280的比率在1.8和2.1之间。琼脂糖凝胶电泳检查mRNA质量和大小。在某些情况下，考虑在转录反应中加入化学修饰的核苷酸。可能需要调整细胞数量、mRNA和脂质体的用量。转染当日理想存活细胞密度在70% ~ 90%之间。有关转染优化技巧，请访问:thermofisher.com/transfectionsupport。

我不慎将我的脂质体试剂留在了室温条件下，还能继续使用吗？

可以，我们所有的脂质体转染试剂均能够在室温下稳定保存数月。

在转染mRNA的过程中，细胞是否会将将外源RNA包裹进囊泡从而降低mRNA的转染效果，还是它们会（部分）滞留在胞浆中？如果被包裹进囊泡，进入囊泡的mRNA和留下可用于翻译过程的mRNA百分比数量分别是多少？如果是DNA质粒的话，又会观察到何种情况？

我们并没有观察到细胞在包裹mRNA的脂质体复合物和DNA的脂质体复合物上有任何差别。转染过程包括脂质体与mRNA之间复合物的形成，随后细胞通过内吞作用吸收上述脂质体复合物。这一过程中脂质体会对mRNA起保护作用，同时辅助其脱离内涵体，从而释放进入细胞胞浆中。此时mRNA可即刻由核糖体进行翻译。mRNA本身可通过如mMESSAGE mMACHINE T7 Ultra转录试剂盒一类的体外转录试剂盒进行制备，后者可向mRNA中加入5’端ARCA帽结构和3’端poly(A)尾，从而良好地模拟了内源mRNA。

何种转染试剂最适合于在低pH值条件下向细胞/组织中转染mRNA？我尝试了市面上绝大多数的转染试剂，结果都不理想。

向细胞中转染mRNA的最佳试剂为Lipofectamine MessengerMAX转染试剂，因为它在更大程度上保护mRNA分子免受降解，进而在广泛细胞种类中显示出最高的mRNA递送效率。请访问此页面（http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/transfection-selection/lipofectamine-messengermax.html?CID=fl-messengermax）获取更多相关信息。对组织或小动物模型而言，最佳试剂应为Invivofectamine 3.0转染试剂，其所采用的纳米技术能够包裹并保护递送的负载物。Invivofectamine 3.0转染试剂既可用于体外细胞转染，也适用于全身性静脉注射法或直接注射（如肌肉、肿瘤、心脏、大脑）。更多详细信息请见此处（http://www.thermofisher.com/us/en/home/life-science/rnai/introduction-to-in-vivo-rnai/invivofectamine-reagent/invivofectamine-reagent-data.html?cid=fl-invivofectamine）。
此外，也可应用化学修饰的核酸合成mRNA，以提升mRNA的稳定性和进一步减少降解。设置一组阳性对照mRNA（如GFP mRNA）对于优化转染技术很有帮助。

转染mRNA的过程是否更快速？标准的孵育时间有多长？

是的，转染mRNA能够带来更迅速的蛋白翻译，因此蛋白表达也更快。对于某些细胞系，我们在转染后90分钟即观察到GFP的表达。此外，使用Lipofectamine MessengerMAX试剂转染mRNA还能够延长表达的时程（转染后GFP表达可延续5天），这是因为该转染操作能够保护mRNA不受降解。

进行mRNA转染操作时，是否需要使用专门的培养基？同时，我是否需要在转染之后更换培养基？

不需要，进行mRNA转染操作时无需使用专门的培养基，也无特殊限制。对于转染方案，我们只是建议用户在脂质与mRNA复合的过程中确保无血清或抗生素的存在。我们自身的转染方案中使用的是Opti-MEM I减血清培养基（Reduced Serum Medium）。转染mRNA后无需更换培养基。通常的实际操作中，我们在转染后的首个24小时之内不更换培养基，这样是为了尽可能减少对细胞的操作。

我们目前使用DNA转染技术；您能介绍一下mRNA转染的技术优势以及这是细胞系特异的吗？

mRNA转染技术相比DNA转染拥有许多优势，而且该技术具有细胞系特异性。

能够实现更高的转染效率：

如果您通过DNA转染方式在难转细胞中效率低于30%，换用mRNA进行转染就可能获得高达80%的转染效率。DNA递送的部分困难在于其转染过程须顺利达成多个步骤。为了成功实现细胞中的蛋白表达，通常情况下DNA需要转染进入细胞胞浆，再入核，转录为mRNA，输出细胞核，再于胞浆内被翻译为蛋白质。直接递送mRNA时，mRNA一进入胞浆即可被翻译为蛋白质。不过，如果您对目前所使用细胞上已达到的转染水平很满意，则无需换用mRNA转染。
无基因组整合风险的无印记方法：

mRNA转染是瞬时性的，不会入核，也不会发生与细胞宿主DNA整合的风险；目前正在探索这项技术未来应用于疫苗置换（vaccine replacement）和疾病模型发展的可能性。
此外，使用全新研发的Lipofectamine MessengerMAX试剂进行mRNA转染可以在更广泛的细胞系（如原代神经元、原代肝细胞、原代成纤维细胞、iPS细胞、hNSC、mESC、Raw 264.7, SH-SY-5Y, HT-29）中获得更好的转染效率。这样就能够不依赖于所使用的细胞模型来递送最高数量的mRNA。

能否使用Lipofectamine MessengerMAX转染试剂递送质粒DNA？

可以，Lipofectamine MessengerMAX转染试剂可用于同时递送质粒DNA和mRNA（在CRISPR技术中）；不过Lipofectamine 3000专为递送质粒DNA进行了条件优化，因此能够达到更好的转染效率。

使用Lipofectamine MessengerMAX转染试剂有多简单？

Lipofectamine MessengerMAX转染试剂拥有极简单的单管实验方案（http://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_MessengerMAX_man.pdf）。用户可购买mMESSAGE mMACHINE ULTRA T7转录试剂盒进行转录，也可直接购买预制的mRNA（如GeneArt CRISPR 核酸酶mRNA，货号A25640）。

我在何处可买到GFP mRNA阳性对照品？

我们公司内部验证了TriLink EGFP mRNA对照（货号L-6101）但由于其已停产，您可购买替代产品Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201)。

MessengerMAX试剂中包含体外转录mRNA用的相关试剂么？

虽然没有，我们推荐使用 mMESSAGE mMACHINE T7 ULTRA Transcription Kit (货号 AM1345)来获得产生加帽和加尾的mRNA以确保最高的mRNA稳定性。

你们建议在何种情况下使用Lipofectamine MessengerMAX转染试剂？

Lipofectamine MessengerMAX转染试剂专用于向细胞转染mRNA。其在神经元和各类原代细胞中具有高达普通DNA转染试剂5倍的转染效率。之所以能够达到如此效果，主要是因为我们拥有了创新性的脂质纳米颗粒技术，该技术将mRNA的递送效果优化至前所未有的高度，同时免去了DNA入核的步骤。这就带来了一个附加的好处：使蛋白表达过程更为迅速并且无基因组整合风险。此外，使用mRNA CRISPR能获得了超出10倍的切割效率。

在向干细胞导入目的基因的操作中，应选择何种转染试剂？

我们建议使用Lipofectamine Stem转染试剂在多种干细胞中递送DNA、mRNA和共转染(siRNA和质粒DNA)。Lipofectamine Stem转染试剂还可提供用于基因编辑的Cas9 - gRNA核糖核苷酸蛋白的导入。有关该试剂转染效率和多功能性的更多信息，请访问:https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-stem-transfection-reagent.html

你们是否提供转染mRNA的转染试剂？

我们提供Lipofectamine MessengerMAX转染试剂来帮助用户转染mRNA，本产品能够在神经元和原代细胞中提供高达5倍的DNA转染效率。

进行转染操作时，你们建议使用何种对照？

为了确保最佳转染成功，我们建议包括阳性转染对照和额外的对照来确认细胞健康和试剂质量。

对于DNA转染，我们建议使用pJTI R4 Exp CMV EmGFP pA载体(货号A14146)。对于siRNA转染，我们建议使用BLOCK-iT AlexaFluor红色荧光对照(货号14750100)或Silencer Select GAPDH阳性对照siRNA (货号4390850)。对于蛋白质转染，我们建议与EmGFP mRNA如Tri-Link CleanCap EGFP mRNA (货号L – 7201)共转染。

细胞健康和试剂质量对照包括：

纯细胞组
细胞+DNA或RNAi组
细胞+脂质试剂组
细胞+GFP质粒阳性对照组

为获得最佳转染效果使用脂质体转染试剂应注意的要点有哪些？

以下要点需要考虑：

1. 针对您的细胞类型来选择阳离子脂质试剂以获得最高转染效率。请参考转染试剂选择指南(http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)进行正确的选择。
2. 优化阳离子脂质试剂和DNA的用量。除细胞状态以外最重要的因素是脂质体：DNA的比率。
3. 在复合物形成过程中不要使用血清。血清中可能含有能一直复合物形成的化合物。我们推荐使用Opti-MEM I减血清培养基以获得最佳的复合物形成效果。然而不含血清的DMEM或PRMI 1640也是可以使用的，但复合物形成效率可能不及Opti-MEM I减血清培养基那样高。
4. 在配制用于DNA-阳离子脂质体复合物形成的培养基中不要使用抗生素、EDTA、柠檬酸盐、磷酸盐、硫酸软骨素、透明质酸、硫酸葡聚糖或其他硫酸蛋白聚糖。
5. 转染时的细胞（密度）应处于60%至80%的汇合度。细胞应处于对数生长中期。为了确保在实验组间获得最为稳定的结果，建议您最好能够在实验开始时优化细胞铺板密度，而不是仅仅通过细胞汇合度来粗略估计。
6. 请确保转染DNA中的启动子-增强子能够兼容靶标细胞类型。
7. 不要使用冰冻过的，或在温度低于4°C的冰箱隔间中储存的阳离子脂质试剂。
8. 请在转染分析实验中加入阳性对照（比如选择货号 A14146 为追DNA转染对照选择货号 14750100 作为siRNA转染对照）。

同时，请参考此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html)列举的要点提示。

你们的转染试剂的稳定性如何？

我们的转染试剂是在常温条件下运输的，在收到产品后应立即置于4℃条件下储存。如果贮存和使用得当，除了管子标签或者产品COA文件中有特别标注外，我们保证从收到产品之日起一年内产品的性能正常。我们不推荐冻存转染试剂，因为冻存一般会降低转染效果。

关于室温运输转染试剂的说明请参见此白皮书(http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf)

为何转染之后我观察到细胞出现毒性反应？你们能提供一些帮助吗？

此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#2)是一些转染后细胞活性降低的可能原因及其相应的解决方案。请注意，最近研究发现，转染过程中可在培养基中使用抗生素。我们对多个细胞系在含与不含抗生素的培养基中的转染效果进行了比较，同时评估其转染效率和细胞毒性，结果显示并无差别。对稳定转染而言，转染操作后至少等待72小时以上，再加入选择性抗生素。

我实验的转染效率很低。你们能给予一些问题排查提示吗？

此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#1)收录了一些转染效率低的可能原因及其相应的解决方案。

如何制作剂量反应曲线或致死曲线？

剂量反应曲线是一种测定细胞接触不同浓度抗生素时的细胞毒性的有力工具。筛选抗性细胞所需的选择性抗生素的量因多种因素而异，包括细胞类型和抗生素类型。我们建议每次制作剂量-反应曲线时使用新的抗生素（或不同的品牌）或是使用不同的细胞系。
剂量-反应曲线测定的实验概要：

1. 将细胞铺在一定数目的孔中，使得它们有25-30％的汇合度。这表明细胞仍在分裂，并对抗生素反应良好。
2. 使用生长培养基稀释待测抗生素至推荐范围的广泛线性浓度。
3. 从细胞中移去生长培养基。将含抗生素的培养基加到各自孔中，留一组孔空置不加。在这些空孔中加入不含抗生素的生长培养基。
4. 在适当的生长条件下培养细胞（每隔3-4天更换一次培养基以去除死细胞，同时添加新鲜的含抗生素培养基）并每日观察细胞状态。在10-14天时，估算每孔中的活细胞数目。（这个时间周期取决于待测抗生素。抗生素如Geneticin、Hygromycin和Zeocin需要大约3周杀死细胞，因此等待10-14天较为理想。而Blasticidin杀死细胞需要大约2周，等待7-10天即可。）为了达到这一点，移去培养基，用磷酸盐缓冲盐水洗涤细胞并用0.5％的亚甲基蓝和50％甲醇进行细胞染色20分钟。
5. 将对应于不同抗生素浓度的活细胞数目绘图。这个曲线即是剂量-响应曲线或致死曲线。抗生素在所选时间段内杀死所有细胞的最低浓度就是接下来用于稳定筛选的浓度。

病毒转导相对于转染的主要优势在哪里？

转染无法用于某些细胞类型，例如非分裂细胞，而病毒转导既能用于分裂细胞又能用于非分裂细胞，比如难以转染的神经细胞。

相对于磷酸钙介导的转染，脂质体介导的转染的主要优势在哪里？

脂质体介导的转染的主要优势是能取得更高的转染效率，即使是不能使用磷酸钙介导转染的细胞类型。此外，脂质体介导的转染不仅能用于寡聚核苷酸到大DNA范围内的DNA递送，还能递送RNA和蛋白质。

什么类型的分子可以使用转染试剂转染？

我们的阳离子脂质体转染试剂可用于转染DNA、siRNA、Stealth RNAi、mRNA、dicer-产生的siRNA混合物、或shRNA质粒。寡核苷酸，蛋白质和RNA也可由其转染。DNA可以是质粒、粘粒，甚至是长达600 kb的YAC克隆。

哪里可找到使用过你们试剂的其他研究者的参考文献？

访问每种类型试剂的产品页面，在页面底部即可看到有一个引用列表。您也可访问一个罗列特定细胞系参考文献的表格。我们还建议采用www.highwire.org作为搜索引擎来查找使用我们转染产品的最新研究论文。您只需在搜索条件中直接列出转染试剂的名称和您的细胞系/应用。

在哪里可以找到特定细胞系的转染方案？

特定细胞系的转染方案可点击此处(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support.html)找到。如果您未找到特定细胞系转染方案或者方案没有达到预期效果，我们建议测试随产品提供的实验方案中描述的条件，确定最佳的实验方案。 成功的转染依赖于细胞类型、脂质体用量、细胞的健康情况、传代次数和转染时的细胞密度。这些因素在各个实验室之间可能略有不同，为了得到相同的结果，可能需要对实验方案进行额外的优化。请访问我们的非常有用的转染实验问题排查技巧页面（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html.），更多关于问题排查的信息欢迎访问我们的转染支持中心(thermofisher.com/transfectionsupport)。

为什么我的基因在瞬时转染的表达水平要高于稳定转染？

瞬时转染克隆的表达水平通常比较高，因为瞬时转染细胞通常具有较高的外源基因拷贝数（每个细胞几百个）。稳定转染的克隆通常有1-2个拷贝整合进基因组，因此其表达水平较低。有时候，稳定转染细胞的表达水平低是因为当组成型表达重组蛋白时对细胞造成了负面影响。

为什么我的转染实验不具有可重复性？

一般情况下，无论如何尝试控制转染影响因素，两次转染的效率仍会表现出一定程度的差异性。转染时，请保持所有转染影响因素，如细胞汇合度、传代次数和生长期一致。尽可能解冻新的细胞。为了尽量减少转染差异的影响，可以使用内参，如β半乳糖苷酶或萤光素酶。可以共转染表达质粒和参考质粒，分析β-gal或荧光素酶的活性。

我使用的孔的大小与您在试验方案中详细描述的有所不同。该如何放大或缩小我的转染体系呢？

我们的每个转染试剂实验方案均提供了一个表格，用于放大或缩小转染体系。详细信息请参考具体的说明书。

我可以针对不同的细胞系使用相同量的任意转染试剂吗？

不可以，转染效率和每个孔所用试剂的量密切相关，并可能因试剂而异。为了获得最佳转染效率，请参考随转染试剂提供的产品信息。
随产品提供的实验方案将为您提供每个孔转染试剂的最佳使用量范围。在产品开发期间，该使用量在多种细胞系中都能很好地工作。如果在您特定的细胞系中没有得到预期的效果，可能还需要做进一步的优化。请访问我们的非常有用的转染实验问题排查技巧页面（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html.），更多关于问题排查的信息欢迎访问我们的转染支持中心(thermofisher.com/transfectionsupport)。

细胞密度（％汇合度）和传代次数是转染需要重要考虑的因素吗？

是的，细胞密度会影响转染性能。 Lipofectamine 3000, Lipofectamine 2000和 Lipofectamine LTX/PLUS能够在70-90％的细胞汇合度时展现优异的转染性能，而汇合度低于这一数值时，则可能观察到细胞毒性。Lipofectamine RNAiMAX在60-80％的细胞汇合度时效果最佳。
传代次数可能会影响转染实验。我们推荐一致地传代和接种细胞。传代次数过多可能会降低转染效率。我们不推荐超过20-30次传代的细胞。如果转染性能下降并且细胞已经培养过很长时间或过度/不当传代，我们建议您从液氮中复苏一瓶新细胞重新培养。请参阅 Gibco细胞培养基础手册(https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html)，了解正确培养和传代细胞的指南。

关于转染试剂的选择，你们有什么建议？

请参阅转染试剂选择指南(https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)进行正确的选择。

转染有哪些不同的方法？

目前有很多种基因递送技术，可向真核细胞中导入质粒DNA、siRNA或双链RNAi、寡核苷酸和RNA，以便进行各种研究和药物开发应用。此处(https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html)提供了有关这些技术及其优缺点的综述。

I am trying to troubleshoot our mRNA transfection process using Lipofectamine MessengerMAX Transfection Reagent. Could you tell me some reasons why we may not be seeing expression after transfection? We are using primary cells, so I am not sure if it is due to the difficulty in transfecting these cells or another cause.

There are reasons that can influence expression after transfection, but before troubleshooting all the possibilities, a transfection experiment with a positive GFP mRNA control (Tri-Link CleanCap EGFP mRNA, Cat. No. L-7201) and the Lipofectamine MessengerMAX mRNA Transfection Reagent could be the solution. If this does not yield good results, it might be best to try an alternative delivery solution or different cells. However, if this gives acceptable results, the next step would be to try the mRNA of interest with the MessengerMAX reagent. If there are expression level concerns at this point, it might be the mRNA that is being used, and troubleshooting from this perspective might be needed. For example, some questions to ask would be: Is there a 5' cap? Is there a poly(A) tail? Is the mRNA pure? Do I get a single band on a gel? Was the DNA template clean?

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

We have seen quite a bit of variation in our mRNA transfection efficiency with Lipofectamine MessengerMAX Transfection Reagent, based on the number of passages prior to transfection. Do you have advice on the best passage number?

It is normal to observe some transfection efficiency differences between cell passages.To minimized this variability, we recommend using cells between 5-20 passages. Including a positive GFP mRNA control (Tri-Link CleanCap EGFP mRNA, Cat. No. L-7201) with every transfection experiment is helpful to quantitatively determine and follow any changes in transfection efficiencies from week to week.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I have been getting some toxicity in my mRNA transfections using Lipofectamine MessengerMAX Transfection Reagent. What would you recommend for minimizing toxicity?

Consider including appropriate 5' UTR and 3' UTR sequences into the template design to promote mRNA stability and acceptance by the cell. Use high purity in vitro transcribed mRNA purified with the MegaClear Transcription Cleanup Kit. Ensure A260/280 ratios are between 1.8 and 2.1. Check for mRNA quality and size by agarose gel electrophoresis. In some cases, consider incorporating chemically modified nucleotides in the transcription reaction. Cell number, mRNA, and lipid quantities may need to be adjusted. Ideal viable cell density on the day of transfection is between 70 and 90% confluence. For transfection optimization tips, please visit: thermofisher.com/transfectionsupport.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I accidentally left my lipid reagent at room temperature. Can I still use it?

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

When transfecting mRNA, does the packaging of the foreign RNA into vesicles reduce the accessibility of the mRNA for protein translation or is the mRNA all released in the cytoplasm? If packaged away, what percent is packaged vs. what percent is left to be available for translation? Is this observed with DNA plasmids?

We have not observed differences between how a cell packages an mRNA payload versus a DNA payload for the purpose of delivery. Transfection involves complex formation between a liposome and mRNA, which create lipoplexes that are taken up by the cell via endocytosis. The liposome protects the mRNA during this process and also assists in endosomal escape, which releases the mRNA into the cytoplasm of the cell. The mRNA is immediately available for translation with the ribosome. In vitro transcribed mRNA may be prepared using the mMESSAGE mMACHINE T7 Ultra Transcription Kit, which incorporates a 5' ARCA cap and a 3' poly(A) tail to mimic endogenously transcribed mRNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the best transfection reagent to use to transfect mRNA into cells/tissue in a low-pH environment? I have tried most of the mRNA transfection reagents on the market, and they do not work.

The best reagent to transfect mRNA into cells is Lipofectamine MessengerMAX Transfection Reagent because it has been shown to further protect the mRNA molecules from degradation and has been shown to deliver the highest amount of mRNA into a wide variety of cell types. Please visit this page (http://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-messengermax.html) for more information. For tissues or small animal models, the best reagent is Invivofectamine 3.0 Transfection Reagent, which is based on a nanoparticle technology that encapsulates and protects the payload for delivery. It can be used on cells in vitro as well as injected systemically via intravenous route or via direct injection (i.e., muscle, tumors, heart, brain). More information can be found here (http://www.thermofisher.com/us/en/home/life-science/rnai/introduction-to-in-vivo-rnai/invivofectamine-reagent/invivofectamine-reagent-data.html?cid=fl-invivofectamine).

In addition, mRNA can also be synthesized with chemically modified nucleotides to improve stability and further minimize degradation. A positive control mRNA (e.g., GFP mRNA) is very helpful to optimize transfection techniques.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is transfection with mRNA faster? What are standard incubation times?

Yes, transfection with mRNA results in faster and more immediate translation of protein and therefore, faster expression. We visually see expression of GFP in some cell lines as fast as 90 minutes after transfection. Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent also provides prolonged duration of expression (GFP expression lasting for 5 days post-transfection), due to its ability to protect mRNA from degradation during transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are there specific medium requirements when using mRNA for transfection? Also, would I have to change the medium after transfection?

No, there are no specific medium requirements or restrictions when using mRNA for transfection. The only recommendation is to ensure that there is no serum or antibiotics present during the complexation process of the lipid and mRNA within the transfection protocol. We use Opti-MEM I Reduced Serum Medium in our transfection protocol. The delivery of mRNA does not require a medium change after transfection. Our common practice is to not change medium within the first 24 hours following transfection, so as to minimize handling of cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

We currently use DNA for transfection, but what are the advantages for mRNA transfection? Is it cell line-specific?

There are many advantages to using mRNA vs. DNA for transfection, and they are cell line-specific:

Higher transfection efficiency:
If you are working with a difficult-to-transfect cell type, where DNA transfection yields less than 30% efficiency, transfecting with mRNA can provide up to 80% transfection efficiency. During DNA transfection, the DNA encounters many hurdles before reaching the nucleus before undergoing the molecular processes that ultimately lead to protein expression. The DNA-lipid complexes first fuse to the membrane, enter the cell by endocytosis, DNA is released from the endosomes and and enters the nucleus. In the nucleus, the DNA is transcribed to mRNA, exported from the nucleus, and is finally translated into a protein in the cytoplasm. During mRNA transfection, the mRNA is ready for immediate translation to protein in the cytoplasm. However, if you are satisfied with the level of transfection achieved for a given cell line, there is no need to switch to mRNA.

Footprint-free method with no risk of genomic integration:
mRNA transfection is transient, does not enter the nucleus, or pose a risk of integrating with the cellular host DNA. mRNA transfection is currently being researched for possible vaccine replacement and disease model development.

Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent provides higher efficiency in a wider range of cell types (e.g., primary neurons, primary hepatocytes, primary keratinocytes, primary fibroblasts, iPS cells, hNSC, mESC, Raw 264.7, SH-SY-5Y, HT-29). This is a result of its ability to deliver the highest amount of mRNA independent of the cell model being used.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine MessengerMAX Transfection Reagent to deliver plasmid DNA?

Yes, Lipofectamine MessengerMAX Transfection Reagent can deliver plasmid DNA in combination with mRNA (i.e., in CRISPR); however, Lipofectamine 3000 Transfection Reagent is best optimized for plasmid DNA delivery and superior DNA transfection performance.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How easy is it to use Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Transfection Reagent has a very simple one-tube protocol (http://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_MessengerMAX_man.pdf). The mRNA can either be transcribed using the mMESSAGE mMACHINE ULTRA T7 Transcription Kit or can be purchased ready-to-use (e.g., GeneArt CRISPR Nuclease mRNA, Cat. No. A25640).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I purchase a GFP mRNA positive control to use with Lipofectamine MessengerMAX Transfection Reagent?

We have validated the TriLink EGFP mRNA Control (Cat. No. L-6101) in-house. As this was discontinued, the alternative is Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Does the Lipofectamine MessengerMAX Transfection Reagent kit include reagents for in vitro transcription of mRNA?

Although it does not, we recommend the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Cat. No. AMB1345) to generate capped and tailed mRNA for highest mRNA stability.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

When do you recommend using the Lipofectamine MessengerMAX Transfection Reagent?

The Lipofectamine MessengerMAX Transfection Reagent is specifically designed for transfection of mRNA into cells. It offers up to 5X the transfection efficiency of DNA transfection reagents in neurons and a broad spectrum of primary cells. This is possible because our novel lipid nanoparticle technology is optimized to deliver the highest amount of mRNA possible without the nuclear entry step that is required with DNA. The added advantage is that protein expression is faster with no risk of genomic integration. Additionally, up to 10X higher cleavage efficiency can be achieved using GeneArt Platinum Cas9 Nuclease for CRISPR gene editing applications.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which transfection reagent should I use for delivering my gene of interest into stem cells?

We recommend using Lipofectamine Stem Transfection Reagent for delivery of DNA, mRNA and co-transfection (siRNA and plasmid DNA) in a wide variety of stem cells. Lipofectamine Stem Transfection Reagent also delivers Cas9-gRNA ribonucleoproteins for gene editing applications. For more information on the transfection efficiency and versatility of the reagent, please visit: https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-stem-transfection-reagent.html

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Do you offer a transfection reagent for transfection of mRNA?

Yes, Lipofectamine MessengerMAX Transfection Reagent is our best reagent for mRNA delivery and shows up to 5Xs the transfection efficiency of DNA transfection reagents in neurons and primary cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What controls do you recommend that I use when performing a transfection?

To ensure optimal transfection success, we recommend including a positive transfection control and additional controls to confirm cell health and reagent quality.

For DNA transfection, we recommend using the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146). For siRNA transfection, we recommend using either the BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or Silencer Select GAPDH Positive Control siRNA (Cat. No. 4390850). For protein transfection, we recommend co-transfecting with an EmGFP mRNA such as the Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Cell health and reagent quality controls:
- Cells only
- Cells + DNA or RNA or protein only
- Cells + lipid reagent only
- Cells + Opti-MEM only
- Cells + positive control

Find additional tips, troubleshooting help, and resources within ourTransfection Support Center.

What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

Here are some points to consider:

1. Select the lipid reagent that is likely to result in highest transfection efficiency for your cell type, payload, and application. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent.
2. Optimize both lipid reagent and DNA quantities. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, but the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be between 50% to 80% confluency at the time of transfection (please refer to specific reagent manual for details). Cells should be in the mid-log growth phase. For better consistency and reproducibility of results between transfection experiments, accurately count your cells with either a hemocytometer or the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Confirm that the promoter and/or enhancer (any gene regulatory sequences) of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagent that has been frozen or stored at temperatures below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

For additional tips ,please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the stability of your transfection reagents?

Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of shipment unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance.

Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine transfection reagents.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why do I see cytotoxicity after performing transfection with Lipofectamine 2000? Can you please help?

Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

  1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
    Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
  2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
    Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
  3. Possible Cause: Cell density was not optimal
    Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
  4. Possible Cause: Complexes were added to cells in serum-free medium
    Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
  5. Possible Cause: Complexes not thoroughly mixed in growth medium
    Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
  6. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
  7. Possible Cause: Antibacterial agents were used in growth medium during transfection
    Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
  8. Possible Cause: Transfection reagent stored improperly
    Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
  9. Possible Cause: Cationic lipid reagent was oxidized
    Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
  10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.


Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

I am getting very low transfection efficiency with Lipofectamine 2000. Can you please provide some troubleshooting tips?

Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:

  1. Possible Cause: Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presence of serum
    Suggested Solution: Use serum-free medium for dilutions of plasmid DNA, siRNA, and transfection reagents. Note: we recommend using Opti-MEM | Reduced Serum Medium (Cat. No. 31985-062)to dilute Lipofectamine 2000 and DNA before complexing.
  2. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
    Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µL) ratio of 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine2000 (µL) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
  3. Possible Cause: Not enough plasmid DNA used for dilution or complex formation
    Suggested Solution: Verify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT DNA Assays Kits (Q33130, Q33120).
  4. Possible Cause: Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
    Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPure Nucleic Acid Purification Kits.
  5. Possible Cause: Cell density was not optimal
    Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
  6. Possible Cause: Complexes were added to cells in serum-free medium
    Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
  7. Possible Cause: Inhibitors were present in medium
    Suggested Solution: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
  8. Possible Cause: Problems with assay used to measure efficiency or expression
    Suggested Solution: Use a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
  9. Possible Cause: Promoter-enhancer on vector is not recognized by the cell type
    Suggested Solution: Verify that the promoter-enhancer on your vector construct is compatible with the target cell type.
  10. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
  11. Possible Cause: Transfection reagent stored improperly
    Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.


    12. Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

How do I perform a dose-response curve or kill curve?

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin, Hygromycin, and Zeocin take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of viral transduction over transfection?

Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of lipid-mediated transfection over calcium phosphate-mediated transfection?

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What types of molecules can be transfected using your lipid-based transfection reagents?

Our cationic lipid transfection reagents are used to transfect DNA (plasmids or oligonucleotides), siRNA (or miRNA), mRNA, or proteins. DNA delivered may be in the form of plasmids, cosmids, or even YAC clones as large as 600 Kb. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I find cell line-specific transfection protocols?

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the transfection does not perform as expected, we recommend optimizing the conditions described in the product manual. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For more troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport).

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why are my transfections not reproducible?

In general, transfection efficiency will show some degree of variability between transfection experiments and among replicates in the same transfection experiment. For better reproducibility, keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. We recommend preparing one master mix of the DNA/lipid complexes for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats. To further minimize the effects of transfection variability on data analysis, consider co-transfecting an internal normalization reference control such as beta-galactosidase or luciferase with the expression plasmid. Below are possible reasons for why your transfection results are not reproducible, along with suggested solutions:

  1. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
  2. Possible Cause: Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
    Suggested Solution: Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details. For well or plates sizes not listed in the scaling table, calculate the total surface area and estimate the -fold difference from the 24-well. Use this -fold difference to adjust for reagent volumes, payload quantities, and seeding densities.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any transfection reagent for different cell lines?

No.The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are cell density (% confluency) and passage number important considerations for transfection?

Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either descreasing or increasing the quantity of complexes added to the culture. We recommend using Lipofectamine 3000 since it shows the best flexibility for variable seeding density without showing cytotoxicity issues and maintains high protein expression. Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX/PLUS provide excellent transfection efficiencies at confluencies between 70 and 90%. Some toxicity may be observed at lower confluencies but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media. Lipofectamine RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What recommendations do you have for selecting a transfection reagent?

Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the different methods available for transfection?

There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I deliver single-stranded oligonucleotides with Lipofectamine MessengerMAX Transfection Reagent?

If you are delivering a single-stranded oligonucleotide on its own, we recommend using Lipofectamine RNAiMAX Transfection Reagent. However, Lipofectamine MessengerMAX Transfection Reagent can be used for genome editing purposes when Cas9 mRNA is co-delivered with a sgRNA and a single-stranded or double-stranded donor template.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I use Lipofectamine MessengerMAX Reagent to deliver both my GeneArt Cas9 mRNA and IVT gRNA together?

Lipofectamine MessengerMAX Reagent may be used for both single gRNA delivery or multiplexing (gRNA for multiple targets) purposes with high transfection efficiency when used with GeneArt Cas9 mRNA. For a detailed protocol, please refer to the GeneArt Cas9 mRNA manual (https://www.thermofisher.com/order/catalog/product/A25640?ICID=search-a25640). We recommend using either the Invitrogen GeneArt Genomic Cleavage Detection Kit (Cat. No. A24732) or Invitrogen GeneArt Genomic Cleavage Selection Kit (Cat. No. A27663) for mutant analysis.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which cell types can be used with Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Reagent demonstrates low toxicity and high transfection efficiency for all cell types (easy or difficult-to-transfect, primary, and stem cells).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

When would I use Lipofectamine MessengerMAX Reagent over other transfection reagents?

Lipofectamine MessengerMAX Regent is an excellent reagent for co-delivery of Invitrogen GeneArt CRISPR Cas9 mRNA with in vitro transcribed gRNA for geneome editing purposes. Lipofectamine MessengerMAX Reagent has the added benefit of flexibly delivering short dsDNA or HDR templates (0.5-1 kb) which can be ordered through the Invitrogen GeneArt Strings services.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the Invitrogen Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Transfection Reagent is an animal origin-free transfection reagent, especially formulated for the delivery of mRNA, small RNA (eg.,CRISPR IVT gRNA, siRNA, or miRNA), and short dsDNA or HDR templates (0.5-1 kb). Lipofectamine MessengerMAX Reagent is an excellent reagent choice for CRISPR-mediated genome editing applications.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.