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View additional product information for Silencer™ Select Human Kinase siRNA Library V4 - FAQs (4397918)
176 product FAQs found
载体介导的技术使您能够：
•实现瞬时或稳定的靶点敲低
•在所有细胞类型中实现RNAi，甚至是难转染、原代和不分裂细胞
•通过siRNA的诱导表达，调控基因抑制效应
•可以筛选稳定表达某个siRNA序列的单一细胞群
•利用组织特异性启动子控制体内基因表达
我们建议进行一个转染试剂对照实验，确定您的细胞是否对转染试剂敏感。此外，您可以尝试其他细胞密度和siRNA浓度以减少转染本身的毒性。
在一些情况下，即使mRNA已经被敲低，蛋白的敲低效果也会受蛋白降解速率等其他变量的影响。另外，相比于mRNA，蛋白可能需要更长的时间才能看到敲低效果。
请参考以下可能的原因和建议：
•您测试了多少个siRNA？有任何敲低效果吗？如果所有siRNA都没有敲低效果（<10%），那么可能是检测方法本身的问题。试试使用其他qRT-PCR assay评估敲低效果。
•qRT-PCRassay的靶位点和siRNA切割位点的相对位置如何？如果两者相差3,000碱基以上，问题可能是其它剪接转录本的存在造成的。
•实验的Ct值是多少？在40循环的qRT-PCR实验中它们应该低于35。
•您确定siRNA转染进入细胞中了吗？我们建议使用一个经过验证的阳性对照siRNA来检查转染效率。
请参考以下可能的原因和建议：
•是检测的mRNA水平吗？最可靠的方法是实时PCR。在一些情况下，即使mRNA已经被敲低，蛋白的敲低效果也可能会受蛋白降解速率等其它变量的影响。
•RNA是如何提取的？检测提取的RNA的质量了吗？应确保RNA没有降解。
•使用阳性对照了吗？这可以帮助确定试剂是否有效以及siRNA是否被正确递送进细胞。请在进行实验的同时使用阳性对照siRNA。
•使用转染对照了吗？被转染细胞的百分比是多少？
•进行不同时间的检测了吗？通常，基因沉默可以早在转染后24小时测定。但是，基因敲低的持续时间和程度取决于所用的细胞类型和siRNA的浓度。
•优化过转染条件吗？您可以尝试使用不同的细胞密度和siRNA浓度。
•您使用了多大浓度的siRNA？我们建议测试 5 nM到100 nM之间的多个浓度。
根据您的目的基因，在转染后数小时即可测定mRNA水平。我们建议在转染48小时后检测mRNA的敲低效果。影响检测时间的因素包括转录活性，信使RNA的降解速度，以及是否存在替代通路。要确定敲低的峰值，最好进行一个时间梯度实验。
我们建议将单链RNA溶解在1X TE缓冲液中（在无RNase条件下制备的10 mM TrisCl, pH 7.5, 0.1 mM EDTA）。该缓冲液有合适的pH值并螯合了可能导致RNA降解的金属离子。无RNase水也可以用于溶解。双链RNA（siRNA）以10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA溶液的冻干粉提供。请将其溶解在适量的无核酸酶的水中使RNA浓度达到20 µM，这将还原其冻干前的缓冲液。
作为干粉它们可以在–20°C保存6个月。
可以使用实时PCR检测mRNA水平。通常在转染后24, 48和72小时做一个时间曲线分析。蛋白水平可以在48, 72和96小时后检测。检测时间根据目的基因的不同会有差异。
我们建议至少使用2个靶向同一基因的siRNA。这将为RNAi数据带来更高可信度。
是的，大多数Silencer Select siRNA在正义链末端带有dTdT突起。重要的是反义链（引导链）的3’末端突起会和目的mRNA的5’末端互补，因此，这些末端突起可以由任意不同的核苷酸组成。
是的，我们有针对整个人类基因组以及不同功能基因家族的预制板形式的Silencer Select 文库。我们还提供定制Silencer Select siRNA文库以及靶向人和小鼠的定制Silencer 文库。
Silencer Select siRNA的平均分子量是13,400 g/mol，1nmol = 13.4 µg。
定制的 Silencer或Silencer Select siRNA（21碱基）可通过此链接(https://www.thermofisher.com/order/custom-genomic-products/tools/sirna/）订购。
更长或更短的siRNA，请电邮oligos@ambion.com咨询。
预设计的Silencer siRNA是使用算法设计的，没有经过功能测试。它们靶向于人、小鼠和大鼠基因，并且设计为靶向所有已知剪接变异体。一旦这些siRNA被购买，我们将提供它们的序列信息。我们保证靶向同一目的基因的3个SilencersiRNA中的2个或2个Silencer Select siRNA的2个可以达到大于70%的基因敲低效果。经验证的siRNA是使用算法设计并经过实验验证其mRNA敲低效果大于70%。它们每一个都保证具有基因沉默效果，对于一些人的基因，有验证过的siRNA。验证信息是可以提供的，一经购买，序列信息也可提供。
该修饰是LNA——锁核苷酸修饰——它可以提高与互补RNA形成的双链的热稳定性和特异性。该修饰的位置是受专利保护的。
Silencer siRNA是我们的第一代siRNA，而Silencer Select siRNA是我们的第二代Ambion siRNA。两者使用不同的算法进行设计，但是都包含一个19核甘酸的核心序列和一个2碱基的3’突起。Silencer Select siRNA还包含一种化学修饰。我们建议尽可能使用Silencer Select siRNA，因为它们的效率、特异性，以及基因敲低能力都更好。
要分析一个特定siRNA序列对基因活性的影响，所导入的siRNA的序列必须是已知的。这就要求在加入合成的siRNA或者携带短发夹RNA（shRNA）的载体之后设计、引入并测定基因的封闭效果。当剪切后的siRNA（d-siRNA）被导入细胞后，它们对于引发RNAi效应尤其有效，因为在这一组序列中通常会有数个有效siRNA序列。但是，目前没有方法得知这一效应是由这组序列中哪个（或哪些）特定序列引起的。
最常用的测定特异性基因敲低的方法是进行实时PCR。在某些情况下，可以使用报告基因系统方便地测定报告基因如β半乳糖苷酶的表达。也可以使用免疫印迹分析比较导入siRNA之前和之后的蛋白表达。
两者的分子结构是相同的：Dicer将长dsRNA特异性剪切为21–23个核苷酸的双链，同时带有siRNA标志性的2碱基突起。两者一个重要的区别是d-siRNA通常包含一组从目的dsRNA全长生成的siRNA，而siRNA通常是指靶向特定目的区域的一个单一序列，并且经常以一个单一寡核苷酸序列或几个寡核苷酸的特定组合的形式被合成。
生成siRNA并导入哺乳动物细胞的三种最常用的方法如下：
•体外转录和剪切
•合成siRNA
•携带RNAi元件的载体
RNAi是一种快速鉴定基因功能的经济有效的方法，并且对于目前测试过的大多数基因都有效。RNAi正在迅速成为敲低目的基因表达的首选方法。RNAi对于研究基因功能，信号通路分析，RNAi机制研究，靶点验证均非常有用，并且显示出巨大的诊断和治疗应用潜力。
定制设计：如果不存在预设计的siRNA，那么我们会用自己的算法根据目的基因的序列或数据库编号（accession number）（靶向转录变异体，非人、小鼠或大鼠的物种）设计一个siRNA。定制设计的siRNA是不适用质量保证条款的。
定制合成：用户提供siRNA序列，我们合成。定制合成的siRNA不适用于质量保证条款。
定制修饰：用户可以提出定制修饰，例如加入荧光标记（FAM, Cy3），定制规格（>50 nmol）或分装的报价咨询。定制修饰的siRNA不适用于质量保证条款。
脱靶效应是指由siRNA通过RNAi机制引起的任何非靶基因的沉默。这种效应既可以来自于siRNA引导链（反义链）也可以来自于传递链（正义链）。
通常，我们建议将品牌匹配实验和对照用的siRNA。Silencer，Stealth和Silencer Select siRNA在长度和修饰方面有一些不同。好的实验设计应该使实验和对照siRNA之间的差异最小化。虽然如此，在没有其他选择的情况下，使用和实验siRNA不同种类的阴性对照仍然可以起到排除脱靶效应的对照作用。
是的，我们提供pSCREEN-iT/lacZ-DEST Gateway载体试剂盒，它可以用于进行任何RNAi敲低效果的评估，无需知道蛋白功能，无需进行免疫印迹或qRT-PCR。该系统使用β半乳糖苷酶活性检测来测定RNAi试剂（Stealth siRNA，Silencer siRNA，shRNA质粒，Dicerpools等）的基因敲低效果。将您的目标基因克隆到试剂盒提供的载体上，然后将其和所测试的RNAi试剂共转染。表达的基因产物将和β半乳糖苷酶融合，从而可以根据β-Gal水平来测定RNAi介导的目的基因的mRNA降解水平。
阴性对照是为了反映siRNA对细胞的效果不是由序列的非特异性引起的。阴性对照应该和您的阳性分子的化学特征（长度、修饰）相匹配，但应该是一条不靶向任何特定基因的非靶向型序列。
是的，我们提供几种可选的阴性和阳性对照。阴性对照通常是通用的非靶向序列，而阳性对照是可用作阳性对照和/或方法验证的报告基因的siRNA。请选择和您的siRNA带有同样修饰的对照（如Silencer Select阴性对照，Stealth阴性对照）。
纯化方法的选择取决于您的实验。请参考下列通用指南：
标准纯化：
•脱盐并使用MALDI-TOF质谱分析
•保证至少80%为全长产品
•推荐用于贴壁细胞系
HPLC:
•HPLC纯化并使用MALDI-TOF和分析级HPLC分析
•保证至少97%为全长产品
•推荐用于电转原代或悬浮细胞系
体内级别纯化方式：
•HPLC纯化，使用MALDI-TOF和分析级HPLC分析，透析脱盐，过滤除菌，并经过内毒素测试
•保证至少97%为全长产品
•适用于使用siRNA进行动物体内实验的研究者
请注意，在所有情况中，退火效率均使用PAGE分析。
请查看下列信息：
(a) Silencer Select: “ThermoFisher Scientific向您担保，如果您针对同一靶基因购买了两条预设计的Silcencer SelectsiRNA，这两条siRNA均能够达到对靶基因的70%或以上的敲低效果。此担保的前提条件是，siRNA的转染浓度必须在≥5 nM，且在转染后48小时测定mRNA水平。建议采用实时定量RT-PCR技术检测mRNA水平，但不做强制性要求。客户还必须提供阳性对照的有效敲低结果，从而证明转染效率是足够的。如果未能检测到我们承诺的敲低水平，而阳性对照的结果是成功的，我们将会免费为您合成一条新的Silencer Select siRNA。此担保不适用于任何换货产品。”
(b) Stealth siRNA: “ThermoFisher Scientifics向您担保，如果您针对同一靶基因购买了三条预设计的Stealth siRNA，这三条独立的非重叠的siRNA之中至少有两条能够对靶基因mRNA的敲低效果达到70%或以上。此担保的前提条件是，siRNA转染的终浓度必须≥20 nM，且在转染后48小时测定mRNA水平。建议采用实时定量RT-PCR技术检测mRNA水平，但并不是强制的。客户还必须证明采用阳性对照siRNA能够充分敲低，从而证明转染效率是足够的。如果未能观测到我们担保的敲低水平，而适当的阳性对照成功敲低，我们将会免费为您合成一条新的StealthsiRNA序列。此担保未延伸至任何换货产品。我们还建议您使用适当的阴性对照（如三种Stealth RNAi阴性对照之中的一种）来标准化mRNA的敲低水平。”
(c) Silencer: “ThermoFisher Scientific向您担保，如果您购买了针对同一靶基因的三条预设计的Silencer siRNA，其中至少有两条能够将培养细胞内的靶标mRNA的表达水平降低70%或以上（在100 nM或更高的siRNA终浓度下进行转染，转染后48小时后测定，试验条件如下所述）。如果三条之中至少有两条未能引起>70%的靶标mRNA敲低效果，ThermoFisher Scientific将会免费一次性更换至多三条预设计的SilencersiRNA。产品更换申请必须在预设计SilencersiRNA发货后一百八十(180)天内提出。您必须遵照良好的实验室操作，而且靶向内源基因的经验证的siRNA（例如，Ambion Silencer GAPDH siRNA对照）对照实验结果确认转染效率最佳。为了评估敲低效果，预处理样品内的靶标mRNA水平必须与采用非靶向对照siRNA（例如Silencer阴性对照1）转染的细胞内的靶标mRNA水平进行比较。我们建议采用TaqMan基因表达检测试剂盒定量mRNA水平。”
我们建议使用以下对照：
•阳性对照siRNA
•阴性对照siRNA
•仅有细胞（无siRNA）的对照
•针对靶基因的多条siRNA
•仅有转染试剂（无siRNA）的对照
是的，如果经过反复转染步骤细胞状态没问题即可。
根据您所靶向的基因，在转染后数小时即可在mRNA水平检测。我们建议在转染48小时后检测mRNA的敲低水平。影响检测时间的因素包括转录活性，信使RNA的降解速度，以及是否存在替代通路。要确定敲低的峰值，最好进行一个时间梯度实验。
反义链（引导链）将与mRNA结合。
siRNA代表“短干扰RNA”。它包含两条互补的19-21碱基的RNA链，并带有TT或dTdT突出末端。更长的dsRNA将触发增强的免疫反应而被降解。突出的末端被认为是在Dicer酶剪切过程中添加的。目标分子的剪切被认为是发生在反义链的中间部位，所以中间位置的碱基需要是保守的。合成的dsRNA分子将通过电转或脂质体转染进入细胞内引发RNAi作用。一旦进入细胞，siRNA的两条链将分开，释放出反义链。反义链在RISC蛋白的辅助下结合到与其互补的mRNA分子上。如果碱基完全配对，则mRNA分子将被降解。
RNAi代表RNA干扰。介导RNAi的分子是经过内源Dicer酶剪切的短双链RNA（dsRNA）寡核苷酸。Dicer酶的剪切产物一开始被叫做短干扰RNA，现在被称为siRNA。RNAi技术利用细胞的天然机制，通过转染siRNA而有效敲低特定基因的表达。诱导RNAi有几条途径：合成的分子，RNAi载体，以及体外剪切。在哺乳动物细胞中，短dsRNA——短干扰RNA——启动对细胞内目标mRNA的特异性降解。在这一过程中，siRNA的反义链成为一个多蛋白复合体或RNA诱导的沉默复合体（RISC）的一部分，该复合体随后找到相应的mRNA并在特异性位点将其剪切。然后，该剪切后的信使RNA被特异性降解，并最终导致蛋白表达的敲低。
如果您仍然想要在文库中有4个siRNA靶向同一靶基因，可以选择从我们的人类Silencer文库中加入一个siRNA。
通常认为每个靶基因至少需要两个有效的siRNA证明某个表型是由目的基因敲低引起而不是因为脱靶效应。由于Silencer Select siRNA设计的算法以及创新的化学修饰降低了脱靶效应而不影响siRNA的性能，Silencer Select siRNA与其它siRNA技术相比能更有效诱导产生特异表型的百分比显著提高。
出货时间依据siRNA数量和制板要求的复杂程度而不同。大部分Ambion siRNA文库订单在订单处理后1-3周内完成。您的特定文库的预计出货时间将和您的报价单一起提供给您。请联系您当地的销售代表或发邮件到rnailibraries@lifetech.com获取更多信息或询价。
对于定制siRNA文库，我们可以提供多种制板选择，我们遇到最多的要求是将每块96孔板的最后1列或2列留为空白，并将靶向同一靶点的不同siRNA置于不同板的同一孔位。空白列便于在最终的转染板内加入任何想要的对照。当靶向同一靶点的不同siRNA被置于不同板的同一孔位时，可以方便使用机械臂或排枪将siRNA混合使用。另外，这种形式有利于qRT-PCR分析。
您的基因列表和定制siRNA信息将严格保密，不会用于除询价或合成您的定制siRNA文库之外的任何目的。
Silencer和Silencer Select siRNA人类基因组siRNA文库套装包括靶向NCBI Entrez基因数据库内所列大部分基因的siRNA，每个基因3个siRNA。这些siRNA通常有库存，因此，这些siRNA的成本和价格比我们专门为您定制的siRNA便宜。要提供询价，我们必须比较您的基因列表和我们库存的siRNA列表。
请联系您的当地销售代表或发邮件到rnailibraries@lifetech.com联系我们。我们将为您提供询价表，精确获知您希望如何构建自己的siRNA文库。您将需要准备提供如下信息：
•您的基因/靶点列表（最好是NCBI Entrez基因ID；RefSeq mRNA登录号也可）
•您选择Silencer Select siRNA，Silencer siRNA (非修饰)，还是Ambion In Vivo siRNA
•每个靶点所需的siRNA数目（通常每个编码基因对应3个siRNA）
•每孔所需的siRNA量：
•Silencer Select siRNA有0.1、0.25、1、2或5 nmol规格
•Silencer siRNA有1、2或5 nmol规格
•您想要的预制板类型
以下是一些使用了Ambion siRNA文库/套装的参考文献。这只是一部分。请联系我们获取最新参考文献名录，请发邮件到rnailibraries@lifetech.com.
Silencer Select siRNA
•Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of Src kinases. Ritzerfeld J, Remmele S, Wang T et al. (2011) Genome Res Jul 27 (available online)
•Identification of protein kinases that control ovarian hormone release by selective siRNAs. Sirotkin AV, Ovcharenko D, Mlyncek M (2010) J Mol Endocrinol 44:45–53.
•Selection of hyperfunctional siRNAs with improved potency and specificity. Wang X, Xiaohui Wang X, Varma RK et al. (2009) Nucleic Acids Res:37: e152.
•Protein kinases controlling PCNA and p53 expression in human ovarian cells. Alexander V Sirotkin AV, Ovcharenko D, Benco A et al. (2009) Funct Integr Genomics 9:185–195.
Silencer siRNA
•Identification of host factors involved in borna disease virus cell entry through a small interfering RNA functional genetic screen. Clemente R, Sisman E, Aza-Blanc P et al. (2010) J Virol 84:3562–3575.
•siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. Grimard V, Massier J, Richter D et al. (2008) J Lipid Res 49:2427–2440.
•Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system. (2009) Inglis KJ, Chereau D, Brigham EF et al. J Biol Chem 284:2598–2602.
•Identification of survival genes in human glioblastoma cells by small interfering RNA screening. Thaker NG, Zhang F, McDonald PR et al. (2009) Mol Pharmacol 76:1246–1255.
•Low-dose arsenic trioxide sensitizes glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone via an Akt-dependent pathway. Bornhauser BC, Bonapace L, Lindholm D et al. (2007) Blood 110:2084–2091.
•Visual screening and analysis for kinase-regulated membrane trafficking pathways that are involved in extensive β-amyloid secretion. Adachi A, Kano F, Saido TC et al. (2009) Genes Cells 14:355–369.
•Identification and characterization of 3-iodothyronamine intracellular transport. Ianculescu AG, Giacomini KM, Scanlan TS (2009) Endocrinology 150:1991–1999.
•High-throughput RNAi screening in vitro: from cell lines to primary cells. Ovcharenko D, Jarvis R, Hunicke-Smith S et al. (2005) RNA 11:985–993.
我们使用一个热封系统将封膜附在Ambion siRNA文库板上。为避免胶水粘在板上，您可能需要在板子仍冷冻时（–20°C或–80°C）打开封膜。
是的。请注意这些盖子没有被标记，所以不需要保持盖子和板配套。
Silencer Select人类基因组文库可以384孔板或96孔板形式购买。两种形式分别包含186块384孔板或738块96孔板。每块384孔板的最后两列为空白，而每块96孔板的最后一列为空白。
我们只需在持续更新注释信息的数据库中运行您的siRNA文库文件，就可提供更新的注释信息。要获得之前购买的siRNA文库更新的注释信息，请联系赛默飞世尔技术支持部门，可发邮件至rnailibraries@lifetech.com并提供您的siRNA数据库的批号。
文件中包含一个带有下列栏目的Excel文件。文件的每行代表一个siRNA。
1. 批号：您的订单有一个唯一的批号。
2. 板ID：印在板上的唯一性条码。条码还以人可读形式印在板的标签上。
3. 样本ID：列于该行的siRNA的唯一批号。
4. 板名称：列于板标签上的可识别板名称。
5. 位置（行-列）：该siRNA在板内的行和列位置。
6. 行：该siRNA在板内的行。
7. 列：该siRNA在板内的列。
8. RefSeq登录号：该siRNA靶标基因的RefSeq数据库编号
9. 基因标志：该RefSeq靶基因相应的NCBI Entrez基因标志
10. 基因全名：该RefSeq靶基因相应的NCBI Entrez基因名称
11. 基因ID：该RefSeq靶基因相应的NCBI Entrez基因ID
12. siRNA ID：代表该siRNA序列的唯一编号。我们使用产品编号（或称货号）指定该siRNA序列的合成量、纯化方式以及siRNA ID。要重新订购某个siRNA，您需要提供产品货号和siRNA ID。
13. siRNA量：每孔的siRNA量，以nmol表示。
14. 目标外显子：该siRNA靶向的外显子。
15. 正义siRNA序列：以 5‘至3’排列的siRNA正义链（传递链）序列。
16. 反义siRNA系列：以5‘至3’排列的siRNA反义链（引导链）序列。
17. 经验证与否：A——是‖表明我们已经使用qRT-PCR验证过该特定siRNA序列并且它通过了我们的基因敲低标准（使用5 nM Silencer Select siRNA时mRNA敲低效果达到80%或更多）。
18. 平均剩余RNA水平：在验证实验中平均的剩余mRNA百分比（如果有数据的话）
19. 正向误差线：平均剩余RNA水平的正向误差线（如果有数据的话）
20. 负向误差线：平均剩余RNA水平的负向误差线（如果有数据的话）
21. 细胞系：验证siRNA所用的细胞系（如果有数据的话）。
板的命名旨在作为人可读的板编号，通常以字母和数字的组合结尾，可用于识别靶向相同靶基因的siRNA的板。我们的所有储存文库，以及很多定制文库，都有靶向同一靶基因的不同siRNA位于不同板的同一孔位。这可以被认为是―ABC‖格式，即某一板内的―A‖ siRNA与另一板内的―B‖ siRNA靶向相同靶基因并按同一顺序排列。
是的，文库的每块板都有一个唯一的条码。没有哪两块板具有相同条码编号，因此我们可以根据给定的条码对样本进行追踪。条码编号将一直列在随文库运送的电子数据文件中。
条码位于板的短边的第12列边上（中央）。该标签包括条码，一个人眼可识别的条码定义和一个板名称，以便于使用者对同一文库内的不同板进行排列。
Silencer Select siRNA文库及其亚库（Silencer Select人类药物靶点基因组，Silencer Select人类药物靶点基因组扩展组，以及Silencer Select人类基因组扩展组）被预装于384孔板内，每孔0.25 nmol siRNA。每块板的最后2列空白，并且靶向同一靶基因的siRNA位于不同板的同一孔位。所有其它预制板Ambion siRNA文库都是每孔预装0.25 nmol siRNA于96孔板内。每块板最后1列空白，并且靶向同一靶基因的siRNAs位于不同板的同一孔位。
Silencer Select 人类基因组siRNA文库V4中2.8%的序列和Silencer人类基因组siRNA文库V3中相同。
Silencer Select人类药物靶点基因组siRNA V4文库靶向9,032个基因，而Silencer Select人类扩展药物靶点基因组siRNAV4文库靶向这些基因以及其“扩展组”，总共10,415个基因，包括转录因子。
Silencer Select 人类基因组siRNA文库V4（货号4397926）包含下列内容：
•Silencer Select人类药物靶点基因组siRNA文库V4，384孔板（货号4397922）。该文库包含27,093个独特siRNA (0.25 nmol)，靶向9,031个人类基因的转录本。共78块板：75块板每块352个siRNA，3块板每块232个siRNA。
•Silencer Select人类药物靶点基因组siRNA扩展组V4，384孔板（货号4397924）。该文库包含4,149个独特siRNAs (0.25 nmol)，靶向1,383个人类基因的转录本。共12块板：9块板每块352个siRNAs，3块板每块327个siRNAs。
•Silencer Select人类基因组siRNA扩展组V4，384孔板（货号4397923）。该文库包含33,510个独特siRNAs (0.25 nmol)，靶向11,170个人类基因的转录本。共96块板：93块板每块352个siRNA，3块板每块258个siRNA。
可以发邮件到rnailibraries@lifetech.com获取目标基因列表。
Silencer Select 人类基因组siRNA文库V4中的siRNA靶向21,584个基因，每个目标基因有3个独立的非重叠的siRNA，总共64,752个siRNA。这些目标基因覆盖了98%的NCBI所列的对应至少一个RefSeq编码转录本的基因。这些siRNA被设计为可以靶向该基因所有当时已知的RefSeq编码转录本。该文库中那些靶向药物靶点的siRNAs根据基因功能分类排列以便对重要的基因亚群进行筛选。
我们提供下列现成的人类Silencer Select siRNA文库：
•基因组
•扩展的药物靶点基因组
•药物靶点基因组激酶
•磷酸酶
•G蛋白偶联受体（GPCR）
•离子通道
•核激素受体
•蛋白酶
我们还提供下列定制生产的预定义的Silencer Select siRNA文库：
•癌症基因组
•转录因子
•DNA损伤反应
•凋亡反应
•表观遗传
•药物靶点
•药物转运体
•细胞周期调控
•膜转运体
•泛素连接
•细胞表面肿瘤抑制因子
对于Silencer siRNA文库，我们提供下列人和小鼠预定义套装：
•药物靶点基因组
•基因组
•激酶
•磷酸酶
我们还提供下列人类Silencer siRNA文库：
•G蛋白偶联受体
•人蛋白酶，离子通道和核激素受体
高效和可重复的转染对于任何siRNA实验都是很关键的。siRNA实验的成败经常取决于siRNA的导入。优化siRNA的导入和转染过程中的细胞活力可以避免最常见的导致基因沉默实验失败的因素。最优的转染条件根据细胞系不同而不同。因此，在进行siRNA文库筛选前优化转染条件对于实现最高的基因沉默水平同时将转染过程的细胞毒性降到最低是必须的。
提前投入时间为您的实验体系找到最好的转染条件可以将siRNA摄入最大化、细胞毒性最小化，从而将转染的不稳定性降低并获得最好的数据质量。一旦您找出了最佳的转染方案，有趣和高产的文库筛选工作就可开始了。
很多研究者发现从他们最初的siRNA文库板获得他们想要重新测试或者进行后续研究的siRNA很不方便。我们的所有储存文库，以及很多定制文库，都将靶向同一靶基因的不同siRNA置于不同板的同一孔位。这使得选取针对某一靶点的siRNA进行后续实验非常容易。同时，Thermo Fisher Scientific可以方便的提供便宜的后续实验所需的siRNA，并可以板或者管的形式提供相同的siRNA或者靶向同一基因的其它siRNA用于后续实验。请联系我们询问关于价格和合成时间的信息。
我们对Silencer Select siRNAs (1, 30, 50, 及100 nM 终浓度)进行预先铺板并将其置于室温过夜。然后第二天在这些板内使用Lipofectamine RNAiMAX转染试剂转染HeLa细胞，对靶基因CSNK2A1仍然获得了大于80%的敲低效果。用户需要对于自己所用的细胞系进行优化，因为基因敲低的程度会不同。
使用siRNA转染哺乳动物细胞的效率根据细胞类型和所用转染试剂的不同而不同。这意味着转染所用的最佳浓度需要根据经验确定。因为Silencer Select siRNA表现出比其它siRNA文库更强的基因沉默能力，我们建议Silencer Select siRNA的使用浓度为常规siRNA技术所用浓度的5至20分之一。在对Hela和U2OS人骨肉瘤细胞进行脂质体转染时，我们发现Silencer Select siRNA在2–10 nM浓度下对目的mRNA的敲低效果可以达到80%以上。
一次基因组范围的筛选需要用到800块96孔板。如果进行3次生物学意义的重复，那么总转染的孔数是：800块板 X 96孔 X 3个重复=230,400孔。
每孔需要加入0.15 µL的Lipofectamine RNAiMAX试剂 = 34,560 µL Lipofectamine RNAiMAX。即一次带有3复孔基因组范围筛选共需要~35 mL的Lipofectamine RNAiMAX试剂。
如需在重悬后重新密封，我们建议使用铝质而非塑料的封口膜。我们曾经使用过下列封膜，并取得了不错的效果：
•Thermo Scientific Nunc Microplate Lids; Thermo Scientific货号250002
•Sealing Films, Axygen Scientific,（货号47734-816），描述：铝封膜。在广泛温度条件下( –80°到97°C)保持微孔板的完美密封。适用于光敏感样本。均匀粘附。
RNA寡聚物易于被操作中引入的外源核酸酶降解。操作这些产品时请戴手套。使用RNase-free试剂，试管，以及带有过滤芯的移液枪头。收到文库后，您的siRNA可以长期存储在非自动除霜冰箱的–20°C或更低温度（寡聚物以干粉形式在室温运输）。
Ambion siRNA试剂以干粉形式在室温运输，在收到后应存储在非自动除霜冰箱的–20°C或更低温度。在这些条件下，siRNA至少可稳定存储3年。如果有必要，siRNA干粉（未开封）可以在4°C保存至少一年。
重悬Ambion siRNA板：
对每个板低速离心(最大速度 4,000 x g)，以使所有内容物在开封前集中到板底。
使用70%乙醇或其它去除RNase污染的溶液如RNaseZap RNase Decontamination Solution（货号AM9780, AM9782, AM9784）擦拭密封膜表面。
1. 刺破或小心揭开封膜。小心不要撕碎封膜。
2. 加入无核酸酶的灭菌水，使用排枪或液体操作系统和无菌枪头，重悬至所需浓度。使用推荐体积的Ambion Nuclease-Free水（不是DEPC水）重悬siRNA至方便的存储浓度。建议重悬为10 μM或更高浓度。但是，2–5 μM的存储液浓度更适合用于高通量转染以及在自动化平台上进行的实验。
3. 小心吹打5次以重悬。将溶液在旋转混合器或摇床上室温混匀70–90分钟。这一额外混匀步骤确保充分重悬。
4. 如有必要，简单离心以使液体集中到板底。
5. （可选）将siRNA分装到一个或更多子板中，以减少冻融次数。
6. 存储于–20°C或更低温度。siRNA可以短期存储于4°C，但是应当注意密封以避免蒸发。
长期存储应将其存储在非自动除霜冰箱的–20°C或更低温度。可以将siRNA短期存储在4°C不超过一星期，但是应注意密封以避免蒸发。siRNA重悬浓度>1μM时可在–20°C保存一年。在开封前融化siRNA板并简单离心以使液体集中于板底。
Ambion siRNA文库以干粉形式在室温运输。尽管干粉siRNA非常稳定，但是我们建议您在使用前都将干粉siRNA存储在–20°C（或更低温度）。
建议不要反复冻融工作或筛选板。您可以将密封良好的板短期保存在4°C不长于一个星期，使用时简单离心以使液体集中于管底。工作和筛选板中的siRNA浓度要远低于存储板，因而更容易在反复冻融中降解。要获得最佳结果，仅当您需要加入转染试剂和细胞进行RNAi筛选时再融化筛选板。
一旦您准备好了所有需要的塑料耗材和稀释剂，就可以将siRNA分装到适合进行RNAi筛选实验的无菌组织培养板中。我们建议您将siRNA以液滴的形式分装到无菌培养板的工作表面，然后立即密封并将板保存在–20°C以降低污染或者过度蒸发的风险。siRNA的蒸发将导致其难以和转染试剂形成复合物从而降低转染效率。如果您在操作中观察到过度蒸发的现象，您可以在分装时降低siRNA浓度，增加每个孔分装的液体的体积从而减少蒸发的影响。在大多数情况下，保证96孔板内有20 μL siRNA或384孔板内有10 μL siRNA就可以满足大部分细胞和转染试剂的要求。
文库储存板通常重悬后浓度>1 µM并存储于–20°C或更低温度。最适的保存浓度是10 μM。
对于Silencer siRNA，我们建议将冻融次数限制在10次以内以获得最佳结果。
对于Silencer Select siRNA，我们建议将冻融次数限制在50次以内以获得最佳结果。
请参阅下述方案在板内重悬Silencer及Silencer Select siRNAs（以干粉形式提供）：
1. 对每个板进行低速离心(最大速度 4,000 x g)，以使所有内容物在开封前集中到板底。
2. 小心开封。为避免胶水粘在板上，您可能需要在板仍然冷冻时（–20°C或–80°C）打开封膜。
3. 加入无核酸酶的灭菌水，使用排枪或液体操作系统和无菌枪头，重悬至所需浓度（通常2–10 μM）。
4. 小心吹打5次以重悬，并在室温至少孵育10分钟以充分溶解。
5. 如有必要，简单离心以使液体集中到板底。
6. 将siRNA分装到一个或多个siRNA工作板中以减少反复冻融。应当注意记录冻融次数。
7. 在存储前将新的无菌封膜（如Axygen货号 PCR-AS-200）封在板上。
8. 存储在非自动除霜冰箱的–20°C或更低温度以进行长期保存。siRNA可以短期存储于4°C，但是应当注意封好以避免蒸发。
我们推荐重悬后的siRNA浓度≥1μM。
储存板是原始的重悬的siRNA，通常订购目的是便于长期储存siRNA文库，其浓度范围为1到10 µM。siRNA工作板是中等浓度的siRNA用于制备siRNA筛选板从而用于细胞培养及siRNA筛选实验。它们的浓度范围一般介于50至200 nM之间。siRNA筛选板通常是含有特定量的siRNA的组织培养板，适用于RNAi筛选方案。它们浓度通常是每小份体积(<20 µL水)中含有0.5–2.5 pmol的siRNA。转染试剂和细胞直接加入筛选板进行反向转染。
我们通过流程控制和经验证的仪器量取寡核苷酸。而且，运输前也会对每个板进行目测检查。
合成正确性：使用MALDI-TOF质谱法测量每个单链RNA寡核苷酸样品的质量，并与计算出的质量进行比较。
退火：使用非变性凝胶电泳对一个退火的siRNA样本进行分析。
我们的siRNA文库与大多数自动化液体处理机器人兼容，包括Tecan、Hamilton和定制修改的机器人。我们的研发团队使用Tecan液体处理器与以下板：
96孔板，Axygen 货号PCR-96-FS-C，96孔全裙边（Full Skirt）PCR微孔板
384孔板，Axygen 货号P-384-120SQ-C，120 µL 384深孔“Diamond Plate”方孔透明微孔板。
有许多的方法可用于准备siRNA板，而且每个用户可能有特定的要求。这些特定的需要可能决定您将用何种方法使用您的siRNA文库。最好先确定您操作文库的策略，并在重悬储存板前回答以下问题：
1. 您会为您的文库准备一个备份吗？注意，这样会需要双倍的冻存空间及塑料制品、枪头和密封锡箔用于备份储存siRNA文库，但是您需要一个备份以防万一。
2. 您会在初次重悬siRNA储存板时制备工作或者筛选板吗？如果不会，请将板紧紧密封。siRNA文库储存液能在–20摄氏度或更低温度的非自动除霜冰箱内长期储存。
3. 如果您在重悬时制备工作或者筛选板，您会制备多少筛选板拷贝？您有足够的冰箱空间储存这些拷贝吗？
4. 在筛选板内您会使用多大终浓度的siRNA？您会使用什么转染试剂？您会使用什么细胞类型？在大多数情况下，对于大多数永生贴壁细胞系（如HeLa和U2OS细胞系），Silencer siRNA文库可以用30 nM筛选，Silencer Select siRNA文库可以用5 nM筛选。为获得最佳效果，应用特定的siRNA在选定的细胞中进行转染优化实验来确定筛选板上siRNA的最终浓度。
Vector technologies allow you to:
Achieve transient or stable target knockdown
Perform RNAi in any cell type, even hard-to-transfect, primary, and non-dividing cells
Regulate gene inhibition with inducible siRNA expression
Select for a pure population of cells stably expressing an siRNA sequence
Control gene expression in vivo with tissue-specific promoters
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We would suggest running a transfection reagent control only to determine if your cells are sensitive to the transfection reagent. Additionally, you can try using different cell densities and siRNA concentrations to diminish any toxic effects from the transfection itself.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
In some cases, knockdown of a protein can be affected by other variables such as protein turnover rate, even though the RNA is knocked down. Additionally, a longer time course may be needed to see an effect on protein compared to mRNA.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Please see the following possibilities and suggestions:
- How many siRNA did you test? Is there any knockdown? If there is no knockdown (<10%) in any of the siRNA, then the assay is likely the problem. Try using a different qRT-PCR assay to assess knockdown.
- What was the positioning of the qRT-PCR assay target site relative to the cut site for the siRNA? If greater than 3,000 bases away, the problem could be alternative splice transcripts.
- What are the Cts for the experiment? They should be below 35 in a 40-cycle qRT-PCR experiment.
- Did you confirm the siRNA got into the cell? We recommend using a validated positive control siRNA to check the transfection efficiency.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Please see the following possibilities and suggestions:
- Were the mRNA levels checked? The most reliable method is real-time PCR. In some cases, knockdown of a protein can be affected by other variables, such as protein turnover rate, even though the RNA is knocked down.
- How is the RNA being isolated? Has the quality of the isolated RNA been checked? Ensure that the RNA has not been degraded.
- Was a positive control used? This can help to determine whether the reagents are working and whether the siRNA was delivered correctly to the cell. Run your experiment in parallel with the positive control siRNA.
- Was a transfection control used? What is the percentage of transfected cells?
- Was a time course used? Generally, gene silencing can be assessed as early as 24 hours posttransfection. However, the duration and level of knockdown are dependent on cell type and concentration of siRNA.
- Was optimization of transfection conditions performed? You can try using different cell densities and siRNA concentrations.
- Which concentration of siRNA did you use? We recommend testing multiple concentrations between 5 nM and 100 nM.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Depending on the gene you are working with, it can be measured at the mRNA level as soon as a few hours after transfection. We recommend assessing the mRNA knockdown 48 hours posttransfection. Factors affect the timing include the transcription activity, the turnover rate for the mRNA, and if there are alternative pathways. To determine the peak knockdown, it is best to perform a time course experiment.
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We recommend dissolving the single stranded RNA in 1X TE buffer (prepared under RNase-free conditions (10 mM TrisCl, pH 7.5, 0.1 mM EDTA). This buffers the pH and chelates metal ions that can contribute to RNA degradation. RNase-free water is also acceptable. Duplex RNA (siRNA) comes lyophilized from 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA. Resuspending in the appropriate amount of nuclease free water to bring the RNA conc. to 20 µM will reconstitute the buffer to the same.
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As a dry pellet they can be stored at -20 degrees C for 6 months.
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Measure mRNA levels by real-time PCR. A typical time course analysis would measure at 24, 48, and 72 hours. Protein analysis may be checked at 48, 72, and 96 hours. This will vary depending on the gene being targeted.
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We suggest using at least 2 siRNA targeting the same gene. This will give greater confidence in RNAi data.
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Yes, most Silencer and Silencer Select siRNAs have dTdT overhangs on the sense strand. The important thing is that the antisense (guide strand) 3' overhang will be complementary to the 5' end of the target mRNA. However, the overhangs could have any nucleotide composition.
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Yes, we have a pre-plated collection available for the entire human genome and for different functional classes as Silencer Select Libraries. We also offer custom Silencer Select library services and/or Silencer Libraries for human and mouse.
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The average molecular weight of the Silencer Select siRNA is 13,400 g/mol where 1nmol = 13.4 µg.
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Custom Silencer or Silencer Select siRNAs (21 bases in length) can be ordered using the following link: https://www.thermofisher.com/order/custom-genomic-products/tools/sirna/
For longer or shorter siRNA, please email oligos@invitrogen.com.
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Pre-designed Silencer siRNA are algorithm designed and have not been functionally tested. They are available for human, mouse and rat genes and designed to target all known splice variants. The sequence information is provided once the siRNA is purchased. We guarantee 2 out of 3 Silencer and 2 out of 2 Silencer Select siRNA targeted to the same gene will give you 70% or greater knockdown. Validated siRNA are algorithm-designed siRNA that have been experimentally verified to knock down mRNA levels by 70% or more. They are individually guaranteed to silence, and available for select human genes. Validation data is available, and sequence information is provided once siRNA is purchased.
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The modification is LNA - locked nucleic acid- which improves thermal stability and specificity of duplexes formed with complementary RNA. The location of this modification is proprietary.
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Silencer siRNAs were our first-generation siRNAs, while Silencer Select siRNAs were our second generation of Invitrogen siRNAs. They were designed using different algorithms, but both contain a 19-nucleotide core sequence plus a 2-nucleotide 3' overhang. Silencer Select siRNAs also contain a chemical modification. We recommend using Silencer Select siRNA whenever possible, as they show enhanced efficacy, specificity, and potency.
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In order to analyze the effects of a specific siRNA sequence on gene activity, the introduced siRNA sequence must be known. This requires the design, introduction, and measurement of gene blocking following the addition of synthetic siRNA oligonucleotides or of a short hairpin RNA (shRNA) sequence in a vector. When diced siRNAs (d-siRNA) are introduced into the cell, they are particularly effective at initiating an RNAi effect because generally there will be several effective siRNA sequences that are part of the pool. However, there currently there is no way to identify the specific sequence(s) in a pool that are responsible for the effect.
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The most common way to measure gene specific knockdown is to perform real-time PCR. In some cases a reporter system that allows easy measurement of a reporter gene, such as beta-galactosidase, may be used. Western blot analysis to compare the level of protein expression before and after the introduction of siRNA may also be employed.
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The structure of the molecules is the same: Dicer specifically cleaves long dsRNA into the 21-23 nucleotide duplexes with a 2-nucleotide overhang that is the hallmark of siRNA. A key difference is that d-siRNA typically contains a pool of siRNA generated from the entire length of a long dsRNA target, whereas siRNA generally refers to a single sequence that is specific to a particular target region, and is often synthesized as a single oligo or a specified combination of several oligos.
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The three most common methods of generating siRNA to introduce into mammalian cells are:
- In vitro transcription and dicing
- Synthetic siRNA
- Vectors carrying an RNAi cassette
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RNAi is a cost-effective method for the rapid identification of gene function, and appears to work well for most genes tested to date. RNAi is rapidly becoming the preferred method for knocking out the expression of targeted genes. RNAi is useful for assigning gene function, signaling pathway analysis, RNAi mechanism studies, target validation, and shows tremendous potential for diagnostics and therapeutics.
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Custom Designed: If a pre-designed siRNA is unavailable, we will use our algorithm to custom design one for you using the target mRNA's nucleotide sequence or accession number (transcript variants, species other than human, mouse, rat). Custom designed siRNAs are not guaranteed.
Custom Synthesized: The customer provides the siRNA sequence, and we synthesize it. Custom synthesized siRNAs are not guaranteed.
Custom Modified: A quote can be requested for custom modifications such as fluorescent labels (FAM, Cy3), custom sizes ( greater than 50 nmol) and aliquotting. Custom modified siRNAs are not guaranteed.
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Off-targeting effects are when any gene-silencing effects are caused by siRNAs on nontarget mRNAs through the RNAi mechanism. They could come from the guide (antisense) or passenger (sense) strand of siRNA.
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Typically, we would recommend matching the experimental and control siRNAs by brand. There are differences in siRNA length and modification between Silencer, Stealth, and Silencer Select sequences. A good experimental design would minimize these variables between experimental and control siRNA. That said, in a bind, using a negative control with a different brand than the experimental siRNA would still help to control for off-target effects.
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Yes, we offer a pSCREEN-iT/lacZ-DEST Gateway Vector Kit which can be used to assess the potency of any RNAi knockdown without knowing protein function, western blotting or qRT-PCR. The system uses a beta-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth siRNA, Silencer siRNA, shRNA-containing plasmid, Dicer pools, etc). Clone your target gene into the vector provided, and co-transfect it along with the knockdown reagent being tested. Expressed genes will be fused to beta-galactosidase, enabling you to use beta-Gal levels to measure the amount of RNAi-induced degradation of the target gene.
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A negative control is meant to reveal sequence-independent effects of siRNA on your cells. It should match the general chemistry of your positive molecule (length, modification) but be made up of a nontargeting sequence that will not target any specific gene.
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Yes, we offer several choices for negative and positive controls. Negative controls are typically universal nontargeting sequences, while positive controls are reporter genes that can be used as positive controls and/or protocol validation. Please select a control that has the same modification as your siRNA (i.e., Silencer Select Negative Control, Stealth Negative Control).
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The purification method will depend on your experiment. Please see our general guidelines below:
Standard purification:
Desalted and analyzed by MALDI-TOF mass spectrometry
Guaranteed to be at least 80% full-length product
Recommended for adherent cell lines
HPLC:
HPLC purified and analyzed by MALDI-TOF and analytical HPLC
Guaranteed to be at least 97% full-length product
Recommended for electroporation of primary or suspension cell lines
In vivo-ready:
HPLC purified, analyzed by MALDI-TOF and analytical HPLC, dialyzed to remove salts, sterile filtered, and endotoxin tested
Guaranteed to be at least 97% full-length product
Recommended to researchers using siRNA in animals
Please note that, in all cases, the efficiency of annealing is analyzed by PAGE.
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Please see the following:
(a) Silencer Select siRNA: “Thermo Fisher Scientific guarantees that when you purchase two Silencer Select Pre-designed siRNA to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?5 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.”
(b) Stealth siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Stealth Pre-designed siRNA to the same target, then at least two of those three independent, nonoverlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?20 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi Negative Controls, to normalize message knockdown.”
(c) Silencer siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Silencer Pre-designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Thermo Fisher Scientific will provide a one-time replacement of up to three Silencer Pre-designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer Pre-designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as Invitrogen Silencer GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer Negative Control #1. We recommend Applied Biosystems TaqMan Gene Expression Assays to quantify mRNA levels.”
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We recommend the following controls:
- Positive control siRNA
- Negative control siRNA
- Cells-only control
- Multiple siRNAs per target
- Transfection reagent alone
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Yes, if the cells are doing fine with the transfection protocol.
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Depending on the gene you are working with, it can be measured at the mRNA level as soon as a few hours after transfection. We recommend assessing the mRNA knockdown 48 hours posttransfection. Factors affecting the timing include the transcription activity, the turnover rate for the messenger, and if there are alternative pathways. To determine the peak knockdown, it is best to perform a time course experiment.
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The antisense (guide) strand will bind to the mRNA.
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siRNA stands for “short interfering RNA”. It consists of two complementary RNA strands 19-21 nucleotides long, with TT or dTdT overhangs. Longer dsRNA will trigger increased immune responses and be degraded. The overhangs are thought to be added by dicer when the molecules are processed. Target cleavage is thought to be in the middle of anti-sense sequences, so the middle bases need to be conserved. The synthetics will be delivered into cells to initiate RNAi via electroporation or lipid delivery. Once delivered, the two strands will separate, releasing the antisense strand. With the aid of a protein, RISC, it binds to a complementary sense sequence on the molecule of mRNA. If the base-pairing is exact, the mRNA is destroyed.
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RNAi stands for RNA interference. The molecules that mediate RNAi are short dsRNA oligonucleotides that are processed internally by an enzyme called Dicer. The Dicer cleavage products were first referred to as short interfering RNA, now known as siRNA. RNAi technology takes advantage of the cell's natural machinery to effectively knock down expression of a gene with transfected siRNA. There are several ways to induce RNAi: synthetic molecules, RNAi vectors, and in vitro dicing. In mammalian cells, short pieces of dsRNA - short interfering RNA- initiate the specific degradation of a targeted cellular mRNA. In this process, the antisense strand of siRNA becomes part of a multiprotein complex, or RNA-induced silencing complex (RISC), which then identifies the corresponding mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, which ultimately results in the loss of protein expression.
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The goal of transfection optimization is to determine the conditions that will provide maximum gene knockdown while maintaining cell viability for the particular cell type. There is no single transfection parameter that by itself
ensures efficient uptake by cells in culture. However, there are several critical variables to consider in systematically addressing the optimal siRNA uptake into cells:
• Health of cultured cells
• Cell type
• Transfection method
• Choice and volume of transfection agent
• Exposure time of cells to transfection agent
• Cell density
• Amount of siRNA
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Transfection efficiency achieved using transfection delivery strategies depends on the type of cell being transfected. Cells types differ in features that are associated with high or low siRNA delivery efficiency and viability. Easy-to-transfect cells are those that are readily transfected by traditional lipid or electroporation methods and result in high delivery of siRNA and cell viability. These are typically adherent cells and have flat growth morphology. Difficult to transfect cells are cell types that are intractable to traditional lipid or electroporation strategies. For example, suspension, primary, neuronal, and stem cells are historically difficult to transfect. In addition, difficult-to-transfect cells may have clustered growth morphology.
We generally don't recommend pooling. However, it is up to the scientist to design the experiment that fits their needs best. The major justification for pooling siRNAs for screening is that it reduces off-target effects. While this was a major issue with first-generation siRNAs that were not chemically modified and may have had targeting 3'UTR and acted like miRNA, it is not an issue with newer generations of siRNAs such as Silencer Select siRNAs. Since Silencer Select siRNAs are designed using advanced bioinformatic algorithms and rules, and also incorporate chemical modification, one can achieve robust and guaranteed KD at low concentration with very minimal off-target effects. Pooling involves de-convolution of siRNAs for repeat or second screening. Also, siRNA pooling increases the chance of false positive hits that correlate poorly with subsequent single siRNA screening results.
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This is cell line and reagent dependent but we have found that this complex is stable. We tested up to 3 hrs and there was no decline in knockdown in HeLa cells.
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This is cell line and reagent dependent but we have found that diluted RNAiMAX reagent is quite stable. We tested up to 2 hrs following transfections into HeLa cells with good knockdown. However, we noticed a difference if the lipid was diluted in a tissue culture plate versus a conical tube, but if the transfections occur within an hour there isn't a problem.
We have pre-plated Silencer Select siRNAs (1, 30, 50 and 100 nM final concentration) and left the plates out at room temperature overnight. These plates were then transfected with RNAiMAX reagent in HeLa cells the next day and greater than 80% knockdown of our target CSNK2A1 was still achieved.
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The optimal transfection agent depends upon the cell type used. That being said, we find that Lipofectamine RNAiMAX transfection agent provides excellent transfection efficiency with low toxicity in most cell types.
Good transfection is absolutely essential for effective target knockdown using siRNA, thus it is important to include a positive control siRNA in each experiment. The positive control siRNA should elicit reproducible, easily measured knockdown and/or phenotype in the cells and assay used in your study. If you see maximal knockdown or a phenotype above/below a pre-determined threshold level with this control, you know that measurements from other siRNAs tested on the same day are reliable. Note that it is important to empirically determine the thresholds for each assay and siRNA control pair that indicate a good transfection.
In siRNA experiments, negative controls are just as important as positive controls for obtaining meaningful data. Always include a set of transfections with an equimolar amount of at least one non-targeting negative control siRNA
(e.g., Invitrogen Silencer Select Negative Control #1) - data from these crucial controls serve as a baseline for evaluation of experimental target knockdown.
Nontransfected or cells-only negative controls are also very useful in siRNA experiments. By comparing expression of a housekeeping gene among cultures that were not transfected and cultures transfected with a non-targeting negative control siRNA, valuable information about the effects of transfection on cell viability can be obtained.
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Identity: the mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass spectrometry and compared to the calculated mass.
Annealing: a sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.
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Even after resuspension in water, stock solutions of Silencer Select siRNAs are remarkably stable to freeze-thaw cycles. We have tested Silencer Select siRNAs at 1, 5, 10, and 100 µM concentrations after 100 freeze thaw cycles, and have seen no loss of siRNA integrity when analyzed by HPLC and no loss in function when transfected into cells at 5 nM with knockdown measured two days later by qRT-PCR.
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Invitrogen siRNA Libraries are dried and are therefore shipped at ambient temperature. Although dried siRNAs are remarkably stable, we recommend that you store the dried siRNAs at -20 degrees C (or lower) until ready for use.
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The siRNAs can be stored at 4 degrees C for short-term uses but care should be taken to seal well to avoid evaporation.
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The working dilution plates should be at a concentration that will allow accurate pipetting into the transfection plates.
For most screening situations, we find that 100 nM siRNA is a convenient working solution concentration. For 96-
well plates, 0.5 pmol siRNA per well in 100 µl final transfection volume gives a 5 nM final siRNA concentration.
Therefore, you would use 5 µl of the 100 nM siRNA working dilution.
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For those researchers who want to re-seal plates after resuspension, we recommend aluminum seals over plastic.
We have used the following seal in the past with good success:
Catalog Number: 47734-816
Supplier: Axygen Scientific
Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions (-80 degrees C to 97 degrees C) and reagents.
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Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term uses but care should
be taken to seal well to avoid evaporation.
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Plates are shipped at ambient temperature. In the US and Canada, we use FedEx to ship these products.
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We use a heat seal system to affix the seals on Invitrogen siRNA Library plates. To prevent the glue sticking to the plate, you may want to remove the seal while the plate is still cold.
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Yes. Please note that these lids are not labeled, so you do not need to worry about trying to keep lids and plates aligned.
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We use Axygen clear 96-well full-skirt PCR microplates. (Cat. No. PCR-96-FS-C; we use sterile versions of this plate.)
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The following human Silencer Select siRNA libraries are available:
Genome
Extended druggable genome
Druggable genome
Kinases
Phosphatases
GPCRs
Ion channels
Nuclear hormone receptors
Proteases
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A Stealth RNAi duplex of interest is available for reorder at a scale of 20 nmole or higher.
1. To re-order Stealth RNAi, go to https://rnaidesigner.invitrogen.com/rnaiexpress/quickOrder.do.
2. Enter the HSS (Human Stealth Select RNAi ) or VHS (Validated Human Stealth Select RNAi) number in the field. (To obtain the HSS or VHS number of your specific Stealth RNAi, refer to the spreadsheet included in the CD-ROM.)
3. Click "Order Online"
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Sequence information for the Stealth RNAi Human Kinase Collection can be provided upon request. Also, when you re-order a Stealth RNAi duplex from the collection you will receive the sequence information with the product documentation.
Stealth RNAi duplexes of interest are available for reorder at a scale of 20 nmole or higher.
1. To re-order Stealth RNAi, go to https://rnaidesigner.invitrogen.com/rnaiexpress/quickOrder.do.
2. Enter the HSS (Human Stealth Select RNAi ) or VHS (Validated Human Stealth Select RNAi) number in the field. (To obtain the HSS or VHS number of your specific Stealth RNAi, refer to the spreadsheet included in the CD-ROM.)
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Our Silencer siRNA library offerings can be found here (https://www.thermofisher.com/us/en/home/life-science/rnai/synthetic-rnai-analysis/sirna-libraries/silencer-sirna-libraries.html).
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Our Silencer Select siRNA library offerings can be found here (https://www.thermofisher.com/us/en/home/life-science/rnai/synthetic-rnai-analysis/sirna-libraries/silencer-select-sirna-libraries.html).
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If you still would like to have 4 siRNAs in your library instead of 3, you have the option of adding 1 siRNA from our Silencer designs for the human library.
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It is generally agreed that you need at least two effective siRNA per target to confirm that a phenotype observed is due to knocking down the intended gene and not due to an off-target effect. Due to the strength of the Silencer Select siRNA design algorithm and the novel chemical modification, which serve to reduce off-target effects without negatively impacting siRNA potency, a significantly higher percentage of Silencer Select siRNAs are effective at eliciting on-target phenotypes as compared to other siRNA technologies.
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Turnaround times vary depending on the number of siRNAs and the complexity of the plating request. Most Invitrogen siRNA library orders are completed within 1-3 weeks of order processing. An estimated turnaround time for your particular library will be provided with your price quote. Please contact your local sales representative or email us at rnailibraries@lifetech.com for more information or a quote.
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For custom siRNA libraries, we can provide a number of plating options. Our most requested format is to have the last 1 or 2 columns empty of each 96-well plate, and to have different siRNAs to the same targets plated in separate plates in the same well location. The empty columns facilitate easy inclusion of any desired control in the final transfection plates. When different siRNAs to the same targets are plated in separate plates in the same well location, the siRNAs can easily be pooled with robotics or a multichannel pipettor. In addition, this type of format facilitates analysis by qRT-PCR.
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Your gene list and custom siRNA information is kept strictly confidential and is not used for any other purpose other than to prepare your price quote or to manufacture your custom siRNA library.
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The Silencer and Silencer Select siRNA Human Genome siRNA Library collections include three siRNAs for most human coding genes listed in the NCBI Entrez Gene database. We generally keep these siRNAs in stock, and therefore, our costs and your prices are lower for these siRNAs than ones that we custom manufacture just for you. To provide a quote, we must compare your gene list to our list of inventoried siRNAs.
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Contact your local sales representative or contact us at rnailibraries@lifetech.com. We will provide you with a Quote Request form to indicate exactly how you would like your siRNA library formatted. You will need to be prepared to provide the following information:
Your gene/target list (preferably by NCBI Entrez Gene ID; RefSeq mRNA Accession Number is also acceptable)
Your choice of Silencer Select siRNA, Silencer siRNA (unmodified), or Invitrogen In Vivo siRNA
The number of siRNAs needed per target (typically 3 for coding genes)
The amount of siRNA per well desired:
Silencer Select siRNAs available as 0.1, 0.25, 1, 2, or 5 nmol
Silencer siRNAs available as 1, 2, or 5 nmol
Your desired plating format
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Below are some citations that reference Invitrogen siRNA libraries/collections. This is only a subset. Please contact us for an updated citation list at rnailibraries@lifetech.com.
Silencer Select siRNA
Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of Src kinases. Ritzerfeld J, Remmele S, Wang T et al. (2011) Genome Res Jul 27 (available online)
Identification of protein kinases that control ovarian hormone release by selective siRNAs. Sirotkin AV, Ovcharenko D, Mlyncek M (2010) J Mol Endocrinol 44:45-53.
Selection of hyperfunctional siRNAs with improved potency and specificity. Wang X, Xiaohui Wang X, Varma RK et al. (2009) Nucleic Acids Res:37: e152.
Protein kinases controlling PCNA and p53 expression in human ovarian cells. Alexander V Sirotkin AV, Ovcharenko D, Benco A et al. (2009) Funct Integr Genomics 9:185-195.
Silencer siRNA
Identification of host factors involved in borna disease virus cell entry through a small interfering RNA functional genetic screen. Clemente R, Sisman E, Aza-Blanc P et al. (2010) J Virol 84:3562-3575.
siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. Grimard V, Massier J, Richter D et al. (2008) J Lipid Res 49:2427-2440.
Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system. (2009) Inglis KJ, Chereau D, Brigham EF et al. J Biol Chem 284:2598-2602.
Identification of survival genes in human glioblastoma cells by small interfering RNA screening. Thaker NG, Zhang F, McDonald PR et al. (2009) Mol Pharmacol 76:1246-1255.
Low-dose arsenic trioxide sensitizes glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone via an Akt-dependent pathway. Bornhauser BC, Bonapace L, Lindholm D et al. (2007) Blood 110:2084-2091.
Visual screening and analysis for kinase-regulated membrane trafficking pathways that are involved in extensive beta-amyloid secretion. Adachi A, Kano F, Saido TC et al. (2009) Genes Cells 14:355-369.
Identification and characterization of 3-iodothyronamine intracellular transport. Ianculescu AG, Giacomini KM, Scanlan TS (2009) Endocrinology 150:1991-1999.
High-throughput RNAi screening in vitro: from cell lines to primary cells. Ovcharenko D, Jarvis R, Hunicke-Smith S et al. (2005) RNA 11:985-993.
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We use a heat-seal system to affix the seals on Invitrogen siRNA library plates. To prevent the glue from sticking to the plate, you may want to remove the seal while the plate is still cold (from the -20 degrees C or -80 degrees C freezer).
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Yes. Please note that these lids are not labeled, so there are no worries about trying to keep lids and plates aligned.
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The Silencer Select Human Genome Library can be purchase in 384-well plates or 96-well plates. There are 186 384-well plates or 738 96-well plates in the respective library formats. Each 384-well plate has the last 2 columns empty, while the 96-well plate has the last column empty.
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We can provide updated annotation information by simply rerunning your siRNA library file against our database of continually updated annotation information. To receive updated annotation information for a previously purchased siRNA library, contact Thermo Fisher Scientific Technical Support at rnailibraries@lifetech.com and provide the Lot Number of your siRNA library.
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An Excel file is provided with the following columns. Each row of the file represents a single siRNA.
1. Lot Number: A unique lot number assigned to your order.
2. Plate ID: The unique plate barcode that is affixed to the plate. This barcode is also written in human readable format on the plate label.
3. Sample ID: The unique lot number of the siRNA listed on that row.
4. Plate Name: The human readable plate name as listed on the plate label.
5. Location (Row-Col): The row and column designation of the siRNA within the plate.
6. Row: The row designation of the siRNA within the plate.
7. Column: The column designation of the siRNA within the plate
8. RefSeq Ascession Number: The RefSeq accession number(s) targeted by the siRNA
9. Gene Symbol: The NCBI Entrez Gene Symbol corresponding to the RefSeq target(s)
10. Full Gene Name: The NCBI Entrez Gene Name corresponding to the RefSeq target(s)
11. Gene ID: The NCBI Entrez Gene ID corresponding to the RefSeq target(s)
12. siRNA ID: A unique identifier representing the siRNA sequence. We use Part Numbers (also called Catalog Numbers) to designate the size and purity option, and the siRNA ID to represent the siRNA sequence. To reorder an siRNA, you will need to provide both a Part Number and siRNA ID.
13. Amount: The amount of siRNA per well, in nmol.
14. Exons(s) Targeted: The exon(s) targeted by the siRNA.
15. Sense siRNA Sequence. The sequence, listed 5' to 3', of the sense (passenger) siRNA strand.
16. Antisense siRNA Sequence: The sequence, listed 5' to 3', of the antisense (guide) siRNA strand.
17. Validated: A - Yes, in this column indicates that we have validated this particular siRNA sequence in house by qRT-PCR and that it has passed our knockdown criteria (80% or better mRNA knockdown at 5 nM for Silencer Select siRNAs).
18. Mean RNA Levels Remaining: The percent of mRNA remaining found in the validation experiments (if applicable).
19. Plus Error Bar: The upper error bar of the Mean RNA Levels Remaining (if applicable).
20. Minus Error Bar: The lower error bar of the Mean RNA Levels Remaining (if applicable).
21. Cell Line: The cell line used for siRNA validation (if applicable).
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The plate names are meant to be used as the human readable plate identifiers and typically end in a combination of letters and numbers that can be used to identify plates of siRNAs to the same targets. All of our stocked libraries, and many custom libraries, are plated with different siRNAs to the same targets provided in separate plates in the same well location. This can be thought of as - ABC? format, as the - A siRNA designs within a plate correspond to - B? siRNA designs to the same targets on separate plates in the same order.
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Yes, each plate of the library is provided with a unique barcode. No two plates have the same barcode identifier, and we can trace back samples on any plate given that barcode. The barcode identifiers are always listed in the electronic data file shipped with each library.
The barcode is located on the short end of the plate by column 12 (centered). This label includes the barcode, a human readable definition of that barcode and a plate name that helps users order plates within a library.
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The Silencer Select Human Genome siRNA Library and its subsets (Silencer Select Human Druggable Genome, Silencer Select Human Druggable Genome Extension Set, and Silencer Select Human Genome Extension Set) are plated in 384-well plates, each at 0.25 nmol siRNA per well. Each plate has the last 2 columns empty, and siRNAs to the same target are plated in different plates in the same well location. All other premade Invitrogen siRNA libraries are plated at 0.25 nmol siRNA per well in 96-well plates. Each plate has the last column empty, and siRNAs to the same target are plated in different plates in the same well location.
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2.8% of sequences in the Silencer Select Human Genome siRNA Library V4 are identical to those in the Silencer Human Genome siRNA Library V3.
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The Silencer Select Human Druggable Genome siRNA V4 siRNA Library targets 9,032 genes, whereas the Silencer Select Human Extended Druggable Genome siRNA V4 siRNA Library targets those genes plus the "Extension Set", for a total of 10,415 genes, including transcription factors.
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The Silencer Select Human Genome siRNA Library V4 (Cat. No. 4397926) contains the following:
- Silencer Select Human Druggable Genome siRNA Library V4, 384-well plates (Cat. No. 4397922). This library contains 27,093 unique siRNAs (0.25 nmol) targeting transcripts from each of 9,031 human genes. Total of 78 plates: 75 plates with 352 siRNAs each, 3 plates with 232 siRNAs each.
- Silencer Select Human Druggable Genome siRNA Extension Set V4, 384-well plates (Cat. No. 4397924). This library contains 4,149 unique siRNAs (0.25 nmol) targeting transcripts from each of 1,383 human genes. Total of 12 plates: 9 plates with 352 siRNAs each, 3 plates with 327 siRNAs each.
- Silencer Select Human Genome siRNA Extension Set V4, 384-well plates (Cat. No. 4397923). This library contains 33,510 unique siRNAs (0.25 nmol) targeting transcripts from each of 11,170 human genes. Total of 96 plates: 93 plates with 352 siRNAs each, 3 plates with 258 siRNAs each.
A list of targets is available upon request through rnailibraries@lifetech.com.
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siRNAs in the Silencer Select Human Genome siRNA Library V4 correspond to each of 21,584 genes, with 3 unique, nonoverlapping siRNAs provided per target, for a total of 64,752 siRNAs. The so called target genes correspond to greater than 98% of genes listed by NCBI that have at least one or more curated RefSeq coding transcripts. The siRNAs were designed to hit all RefSeq coding transcripts of that gene that were known at the time of design. The siRNAs targeting the druggable portion of this library are arranged by gene functional class to enable easy screening of important gene subsets.
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The following human Silencer Select siRNA libraries are available off the shelf:
- Genome
- Extended Druggable Genome
- Druggable Genome Kinases
- Phosphatases
- GPCRs
- Ion Channels
- Nuclear Hormone Receptors
- Proteases
We also provide the following predefined Silencer Select siRNA libraries as made-to-order from inventory:
- Cancer Genome
- Transcription Factors
- DNA Damage Response
- Apoptosis Response
- Epigenetics
- Drug Targets
- Drug Transporters
- Cell Cycle Regulation
- Membrane trafficking Transporter
- Ubiquitin Conjugation
- Tumor Suppressor Cell Surface
For our Silencer siRNA libraries, we offer human and mouse pre-defined sets for:
- Druggable Genome
- Genome
- Kinase
- Phosphatase
We also provide the following human Silencer siRNA libraries:
- G protein coupled receptors
- Human proteases, ion channels and nuclear hormone receptors
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Efficient and reproducible transfection is critical for any siRNA experiment. Success or failure of a siRNA experiment often hinges on siRNA delivery. Optimizing siRNA delivery and cell viability during transfection eliminates the most common causes of unsuccessful gene silencing experiments. Optimal transfection conditions vary depending on cell line. As a result, it is imperative to optimize transfection conditions for your cells before screening a siRNA library to empirically determine the highest levels of gene silencing while minimizing toxicity associated with the transfection process.
Investing the time up front to identify the best transfection conditions for your experimental system will limit transfection variability and maximize data quality by maximizing siRNA uptake and minimizing cell toxicity. Once you have identified an optimized transfection protocol, the interesting and productive work of library screening can begin.
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Most researchers find it inconvenient to cherry-pick siRNAs from their initial siRNA library plates to obtain siRNAs that they want to retest or otherwise follow up on. All of our stocked libraries, and many custom libraries, are plated with different siRNAs to the same targets provided in separate plates in the same well location. This makes it very convenient to collect siRNAs for each target easily for follow-up experiments. Also, Thermo Fisher Scientific can readily provide you with cost-effective follow-up siRNAs for your screen and provide identical siRNAs, or additional siRNAs to the same target, in either plates or tubes. Contact us for pricing and turnaround time information.
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We have pre-plated Silencer Select siRNAs (1, 30, 50, and 100 nM final concentration) and left the plates out at room temperature overnight. These plates were then transfected with Lipofectamine RNAiMAX reagent in HeLa cells the next day, and greater than 80% knockdown of our target CSNK2A1 was still achieved. Customers will need to optimize for their cell line, as the level of knockdown will differ.
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The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. This means that the optimal concentration used for transfections should be determined empirically. Since Silencer Select siRNAs exhibit superior silencing potency compared to other siRNAs, we suggest starting concentrations of 5- to 20-fold less than typically used for transfection of your experimental cell lines. We have found that Silencer Select siRNAs reduced mRNA levels >80% at final concentrations of 2-10 nM using lipid-mediated transfection in HeLa and U2OS human osteosarcoma cells.
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It is expected that 800 plates of 96 wells would be used for a genome-wide screen. Allowing for 3 biological replicates, the total number of wells to be transfected are: 800 plates x 96 wells x 3 replicates = 230,400 wells.
0.15 µL of Lipofectamine RNAiMAX reagent needs to be added per well = 34,560 µL of Lipofectamine RNAiMAX reagent= approximately 35 mL total of Lipofectamine RNAiMAX reagent for a single genome-wide screen with 3 replicates.
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For those who want to re-seal plates after resuspension, we recommend aluminum seals over plastic. We have used the following seal in the past with good success:
- Thermo Scientific Nunc Microplate Lids; Thermo Scientific Cat. No. 250002
- Sealing Films, Axygen Scientific, (Cat. No. 47734-816), Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions ( -80° to 97 degrees C) and reagents. Ideal for light-sensitive samples. Uniformly applied adhesive.
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RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when handling this product. Use RNase-free reagents, tubes, and barrier pipette tips. Upon receipt, your siRNAs may be safely stored in a non-frost-free freezer at or below -20 degrees C (dried oligonucleotides are shipped at ambient temperature). Invitrogen siRNA reagents are shipped as dry pellets at ambient temperature and should be stored at -20 degrees C upon arrival in a manual defrost or noncycling freezer. Under these conditions, the siRNAs are stable for at least 3 years. If necessary siRNAs as dry pellets (unopened) can be stored at 4 degrees C for at least a year.
To resuspend Invitrogen siRNAs provided in plates:
Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
Wipe the adhesive foil cover with 70% ethanol or other RNase-decontamination solution such as RNaseZap RNase Decontamination Solution (Cat. No. AM9780, AM9782, AM9784).
1. Thermo Scientific or carefully peel back the foil seal to gain access to wells. Use caution and avoid shredding the seal.
2. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration. Resuspend siRNAs to a convenient stock concentration using the recommended volume of Invitrogen Nuclease-Free Water (not DEPC-treated). Concentrated stocks of 10 µM or more are recommended. However, stock solutions of 2-5 µM may better accommodate dilution schemes for high?throughput transfections and assays conducted on robotic platforms.
3. Gently pipet up and down 5 times to resuspend. Place the solution on an orbital mixer/shaker for 70-90 minutes at room temperature. This additional mixing results in more reliable resuspension.
4. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
5. (Optional) Aliquot the siRNAs into one or more daughter plates, to limit the number of freeze/thaw cycles to which the siRNAs are subjected.
6. Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.
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Store in non-frost-free freezer at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use up to a week, but care should be taken to seal well to avoid evaporation. siRNA libraries can be stored at -20 degrees C as resuspended siRNA at less than or equal to 1 µM for one year. Thaw siRNA plates on the benchtop and centrifuge briefly to collect all liquid prior to opening seals.
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Invitrogen siRNA libraries are shipped dried and are therefore shipped at ambient temperature. Although dried siRNAs are remarkably stable, we recommend that you store the dried siRNAs at -20 degrees C (or lower) until ready for use.
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It is recommended that working or screening plates are not subjected multiple freeze/thaw cycles. You can temporarily store well-sealed working plates at 4 degrees C for up to 1 week, spin plates down briefly to collect liquid in the bottom of the tubes. Working and screening siRNA in are typically plated in much lower concentrations than the stock siRNA plates and are more susceptible to degradation under freeze/thaw conditions. For best results, only thaw screening plates when you are ready to add transfection reagents and cells for RNAi screening protocols.
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Once you have assembled all the necessary plasticware and diluents, the siRNA can be distributed into sterile tissue culture plates suitable for RNAi screening protocols. We recommend that you aliquot the siRNA as a droplet onto the working surface of the sterile tissue culture plate, then seal and freeze the plates at -20 degrees C immediately to reduce risk of contamination or excessive evaporation. siRNA that evaporate are more difficult to complex with most transfection reagents which can lead to decreased transfection efficiencies. You can aliquot larger volume with less concentrated working stock if excessive evaporation is observed in your operation. In most cases, up to 20 µL of siRNA for a 96-well plate, or 10 µL of siRNA for a 384-well plate, is still compatible with a wide variety of cells and transfection reagents.
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Library stock plates are usually resuspended at less than or equal to 1 µM and stored at -20 degrees C or lower. Optimal storage is achieved at 10 µM stock concentrations.
For Silencer siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 10 for best results. For Silencer Select siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 50 for best results.
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Please see the protocol below to resuspend Silencer and Silencer Select siRNAs (provided as dried powder) in plates:
1. Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
2. Remove seal carefully. To prevent the glue to stick to the plate, you may want to remove the seal while the plate is still cold (from the -20 degrees C or -80 degrees C).
3. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration (typically 2-10 µM).
4. Gently pipet up and down 5 times to resuspend, and incubate at room temperature for at least 10 minutes to resuspend completely.
5. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
6. Aliquot the siRNAs into one or more siRNA working plates to limit the number of freeze/thaw cycles to which the siRNAs are subjected. Care should be taken to track the number of freeze/thaw cycles.
7. Place a new sterile seal (such as Axygen Cat. No. PCR-AS-200) on the plate before storing.
8. Store in a non-frost-free freezer at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.
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We recommend resuspending your siRNAs at greater than or equal to 1 µM.
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A stock plate is the original resuspended siRNA, usually ordered for long-term storage of siRNA libraries, in the concentration of <1-10 µM. siRNA working plates are of intermediate concentration of siRNA used to stamp out screening siRNA plates for cell culture and siRNA screening procedures. They are usually in concentrations between 50-200 nM. siRNA screening plates are typically tissue culture plates containing a specified amount of siRNA appropriate for RNAi screening protocols. They are usually in the concentration of 0.5-2.5 pmol of siRNA in a small volume (<20 µL of water). Transfection reagents and cells are directly added to screening plates for reverse transfection procedures.
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We quantify and aliquot our oligos using controlled processes and validated instrumentation. Every plate also undergoes a visual inspection prior to shipment.
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Identity: The mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass spectrometry and compared to the calculated mass.
Annealing: A sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.
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Our siRNA libraries are compatible with most automated liquid handling robots including Tecan, Hamilton, and custom-modified robots. Our R&D teams use Tecan liquid handlers with the following plates:
- 96-well plates, Axygen Cat. No. PCR-96-FS-C, 96 Well Full Skirt PCR Microplates
- 384-well plates, Axygen Cat. No. P-384-120SQ-C, 120 µL 384 Deep Well "Diamond Plate" Microplate with Square Wells, Clear.
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There are many acceptable methods for preparing your siRNA plates, and each user may have unique needs. These unique needs should determine how you work up your siRNA library. It is best to have your strategy for library handling worked out, and answers to the following questions in hand prior to resuspending the stock plates:
1. Will you create a backup copy of your library? Keep in mind that this approach will double the freezer storage space and plasticware, tips, and foil seals needed for the backup stock siRNA library, but you will have a backup in case of need.
2. Will you generate working or screening plates at the time of initial resuspension of the siRNA stock plates? If not, tightly seal all stock plates. The stock siRNA libraries can be stored long term in a non-frost-free freezer at -20 degrees C or lower.
3. If you will be generating working or screening plates at the time of resuspension, how many screening plate copies will you generate? Do you have the appropriate freezer space to accommodate these copies?
4. What final concentration of siRNA will you be using in your siRNA screening plates? What transfection reagent will you use? What cell type will you use? In most cases, Silencer siRNA libraries can be screened at 30 nM and Silencer Select siRNA libraries can be screened at 5 nM for most commonly available immortalized adherent cell lines (like HeLa and U2OS cell lines). For best results, the final concentration of your siRNA in your screening plates should be determined empirically through transfection optimization experiments in your cell types of choice using selected siRNAs for this purpose.
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