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View additional product information for Silencer™ siRNA Transfection II Kit - FAQs (AM1631)
66 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。
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。
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。
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
As a dry pellet they can be stored at -20 degrees C for 6 months.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We suggest using at least 2 siRNA targeting the same gene. This will give greater confidence in RNAi data.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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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Reverse transfection saves time and often results in higher transfection efficiency with siPORT NeoFX transfection agent. However, we currently recommend the traditional transfection method if using siPORT Amine transfection agent. Reverse transfection can be done with siPORT Amine transfection agent, but our scientists found that many cell types were transfected with higher efficiency via the traditional protocol.
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The GAPDH siRNA is validated for use with human, mouse or rat cells. Conversely, the Negative Control siRNA included was designed to have no significant homology to any known human, mouse or rat sequence.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.