使用siRNA进行基因沉默是分子生物学应用之中的一种快速演化工具

There are several methods for preparing siRNA, such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. Irrespective of which method one uses, the first step in designing a siRNA is to choose the siRNA target site. The guidelines below for choosing siRNA target sites are based on both the current literature, and on empirical observations by scientists at Ambion. Using these guidelines, approximately half of all siRNAs yield >50% reduction in target mRNA levels.

制备siRNA有多种方法，比如化学合成、体外转录、siRNA表达载体以及PCR表达组件等。但无论使用哪种方法，设计siRNA的第一步都是选择siRNA靶位点。这里提到的siRNA靶向位点选择指导准则是基于现有文献以及Ambion的科学家的经验观察所总结的。根据该指导准则进行操作，大约半数的siRNA会使得目标mRNA水平降低50%以上。

通过专利的siRNA设计流程，Ambion已经针对超过35,000个基因（人、小鼠及大鼠）设计了siRNA。关于这些高效siRNA的更多信息，请访问我们的Silencer Select siRNA信息页面。如要订购Silencer Select Pre-designed or Validated siRNA，可以先在我们的siRNA数据库中进行搜索。Silencer Select Pre-designed or Validated siRNA可确保沉默效果，且仅由Ambion/Applied Biosystems提供。

If you prefer to design your own siRNAs, you can choose siRNA target sites in a variety of different organisms based on the following guidelines. Corresponding siRNAs can then be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product.

1. Find 21 nt sequences in the target mRNA that begin with an AA dinucleotide.

Beginning with the AUG start codon of your transcript, scan for AA dinucleotide sequences. Record each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites.

This strategy for choosing siRNA target sites is based on the observation by Elbashir et al. (1) that siRNAs with 3' overhanging UU dinucleotides are the most effective. This is also compatible with using RNA pol III to transcribe hairpin siRNAs because RNA pol III terminates transcription at 4-6 nucleotide poly(T) tracts creating RNA molecules with a short poly(U) tail.

In Elbashir's and subsequent publications, siRNAs with other 3' terminal dinucleotide overhangs have been shown to effectively induce RNAi. If desired, you may modify this target site selection strategy to design siRNAs with other dinucleotide overhangs, but it is recommended that you avoid G residues in the overhang because of the potential for the siRNA to be cleaved by RNase at single-stranded G residues.

2. Select 2-4 target sequences.

Our research has found that typically more than half of randomly designed siRNAs provide at least a 50% reduction in target mRNA levels and approximately 1 of 4 siRNAs provide a 75-95% reduction. Choose target sites from among the sequences identified in Step 1 based on the following guidelines:

• Ambion researchers find that siRNAs with 30-50% GC content are more active than those with a higher G/C content.
• Since a 4-6 nucleotide poly(T) tract acts as a termination signal for RNA pol III, avoid stretches of > 4 T's or A's in the target sequence when designing sequences to be expressed from an RNA pol III promoter.
• Since some regions of mRNA may be either highly structured or bound by regulatory proteins, we generally select siRNA target sites at different positions along the length of the gene sequence. We have not seen any correlation between the position of target sites on the mRNA and siRNA potency.
• Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences. We suggest using BLAST, which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST.

3. Design appropriate controls.

A complete siRNA experiment should include a number of controls to ensure the validity of the data. The editors of Nature Cell Biology have recommended several controls (2). Two of these controls are:

• A negative control siRNA with the same nucleotide composition as your siRNA but which lacks significant sequence homology to the genome. To design a negative control siRNA, scramble the nucleotide sequence of the gene-specific siRNA and conduct a search to make sure it lacks homology to any other gene.
• Additional siRNA sequences targeting the same mRNA. Perhaps the best way to ensure confidence in RNAi data is to perform experiments, using a single siRNA at a time, with two or more different siRNAs targeting the same gene. Prior to these experiments, each siRNA should be tested to ensure that it reduces target gene expression by comparable levels.

如果您希望设计自己的siRNA，可根据以下指导准则从不同物种中选择siRNA靶向位点。然后，通过化学合成、体外转录、载体或PCR产物表达等方式进行制备相应的siRNA。

1. 在靶向mRNA序列中找到一段21 nt的以AA二核苷酸起始的序列。

从您的转录本的AUG起始密码子开始，扫描AA二核苷酸序列。记录下每一个AA及之后3’相邻的19个核苷酸作为潜在的siRNA靶向位点。

这种选择siRNA靶向位点的方式是基于Elbashir等人的观察 (1) 设计的，他们发现，3’端带有UU二核苷酸悬臂的siRNA最为有效。该方式同样与使用RNA pol III来转录发卡siRNA相符，因为RNA pol III会在4-6个核苷酸poly(T)处终止转录，从而得到带有短poly(U)尾的RNA。

在Elbashir的论文及后续发表的论文中，带有其他3’端终止双核苷酸突出的siRNA已被证明可以有效诱导RNAi。若需要，您还可以对这种靶向位点选择方式进行修改，以设计带有其他双核苷酸突出的siRNA，不过一般建议避免选择GG突出末端，因为siRNA在单链G残基处可能被RNase剪切。

2. 选择2-4个靶向序列

Ambion研究发现，通常超过一半的随机设计siRNA可以使得靶向的mRNA在RNA水平上至少降低50%，大约四分之一的siRNA则会得到75-95%的降低。按照如下指南对第一步鉴定得到的序列进行靶向位点选择：

• Ambion的研究人员发现GC含量在30-50%的siRNA比G/C含量更高的siRNA活性高。
• 由于4-6个核苷酸poly(T)会成为RNA pol III的终止信号，如果需要用RNA pol III启动来进行表达，要避免4个以上T或A连续出现在目标序列中。
• 由于mRNA的部分区域可能具有较复杂的结构或与调控蛋白质相结合，我们通常会在基因序列的不同位置来选择siRNA靶向位点。我们并未观察到mRNA上靶向位点的位置与siRNA效能之间的相关性。
• 将潜在的靶向位点与相应的基因组数据库（人、小鼠、大鼠等）进行对比，如果靶向序列与其他基因编码序列具有超过16-17个连续碱基对同源性，就不能选择这样的序列。我们建议使用BLAST（请访问NCBI服务器：www.ncbi.nlm.nih.gov/BLAST）。

3. 设计恰当的对照

完整的siRNA实验应当包含一系列对照，以确保数据的有效性。Nature Cell Biology的编辑推荐了几种对照（2）。其中2种对照为：

• 阴性对照siRNA，与您的siRNA具有相同的核苷酸成分，但与基因组无明显的序列同源性。 要设计阴性对照siRNA，可以将基因特异性siRNA的核苷酸序列打乱，然后进行搜索以确保其与任意基因均不具有同源性。
• 靶向相同mRNA的其他siRNA序列。 确保RNAi数据可信度的最佳方法可能就是每次使用一条单独siRNA，对同一条基因使用两条或更多不同siRNA进行实验。在这些实验之前，需要对每条siRNA进行测试以确保它们能够将目标基因的表达水平降到比较接近的水平。

Researchers who initially reported the use of siRNA expression vectors to induce RNAi had different design criteria for their inserts encoding the expressed siRNA. Most of the designs had two inverted repeats separated by a short spacer sequence and ended with a string of T's that served as a transcription termination site. These designs produce an RNA transcript that is predicted to fold into a short hairpin siRNA as shown in Figure 1. The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, vary among different reports (3-11).

Figure 1. Schematic of a typical hairpin siRNA produced by an siRNA expression vector or an siRNA expression cassette and its relationship to the RNA target sequence.

Recommended procedure for siRNA hairpin design

The following recommendations for siRNA hairpin design and cloning strategy are made based on research by our scientists. The first step in designing an appropriate insert is to choose the siRNA target site by following the steps described under "General Design Guidelines" above.

For screening, we typically test four siRNA sequences per target, spacing the siRNA sequences down the length of the gene sequence to reduce the chances of targeting a region of the mRNA that is either highly structured or bound by regulatory proteins. Because constructing and testing four siRNA expression plasmids per target is time-consuming, we find it much easier to screen potential siRNA sequences using PCR-derived siRNA expression cassettes (SECs). SECs are PCR products that include promoter and terminator sequences flanking a hairpin siRNA template. This screening strategy also permits the rapid identification of the best combination of promoter and siRNA sequence in the experimental system. SECs found to effectively elicit gene silencing can be readily cloned into a vector for long term studies. Our scientists have determined that sequences that function well as transfected siRNAs also function well as siRNAs that are expressed in vivo. The only exception is that siRNA sequences to be expressed in vivo should not contain a run of 4 or 5 A's or T's, as these can act as termination sites for Polymerase III.

For traditional cloning into pSilencer vectors, two DNA oligonucleotides that encode the chosen siRNA sequence are designed for insertion into the vector (Figures 2 and 3). In general, the DNA oligonucleotides consist of a 19-nucleotide sense siRNA sequence linked to its reverse complementary antisense siRNA sequence by a short spacer. Our scientists have successfully used a 9-nucleotide spacer (TTCAAGAGA), although other spacers can be designed. 5-6 T's are added to the 3' end of the oligonucleotide. In addition, for cloning into the pSilencer 1.0-U6 vector, nucleotide overhangs to the EcoR I and Apa I restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 2). In contrast, for cloning into the pSilencer 2.0-U6, 2.1-U6, 3.0-H1, or 3.1-H1 vectors, nucleotide overhangs with BamH I and Hind III restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 3). The resulting RNA transcript is expected to fold back and form a stem-loop structure comprising a 19 bp stem and 9 nt loop with 2-3 U's at the 3' end (Figure 1).

Figure 2. Insert design for pSilencer 1.0-U6. This insert is specific for the pSilencer 1.0-U6 Vector and contains the appropriate 3' overhangs for directional cloning into this vector. The loop sequence and length can be varied as desired.

Figure 3 . Insert design for pSilencer 2.0-U6 and pSilencer 3.0-H1. The insert design is specific for the pSilencer 2.0-U6, 2.1-U6, 3.0-H1 and 3.1-H1 Expression Vectors and contains the appropriate overhanging 5' ends for directional cloning into these plasmids. As with pSilencer 1.0-U6 shown in Figure 2, early indications suggest that a great deal of latitude is possible in the design of the loop; here we provide one loop sequence that we find works well.

For cloning into the pSilencer adeno 1.0-CMV vector, DNA oligonucleotides with stem-loop structures are created similar to those of pSilencer 2.0 and 3.0 vectors described above. However, one notable exception is the absence of 5-6 T's from the 3'-end of the oligonucleotides for the CMV-based vector system since the transcription termination signal for the CMV-based vector system is provided by the SV40 polyA terminator. In addition, for cloning into the pSilencer adeno 1.0-CMV vector, nucleotide overhangs containing the Xho I and Spe I restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 4). However, for cloning into the pSilencer 4.1-CMV vector, nucleotide overhangs containing the Bam H1 and Hind III restriction sites are added to the 5' and 3' end of the DNA oligonucleotides, respectively (Figure 5).

Figure 4. Insert design for pSilencer adeno 1.0-CMV Vector. This insert design is specific for the pSilencer adeno 1.0-CMV vector and contains the appropriate overhangs for directional cloning into this vector. The loop sequence and length can be varied as desired.

Figure 5. Insert design for pSilencer 4.1-CMV Vector. This insert design is specific for the pSilencer 4.1-CMV vector and contains the appropriate overhangs for directional cloning into this vector. The loop sequence and length can be varied as desired.

Selection of siRNA targets

In addition to the our own proprietary algorithm and our suggested procedure for selecting siRNA targets by scanning a mRNA sequence for AA dinucleotides and recording the 19 nucleotides immediately downstream of the AA, two other methods have been employed by other researchers. In the first method, the selection of the siRNA target sequence is purely empirically determined (4), as long as the target sequence starts with GG and does not share significant sequence homology with other genes as analyzed by BLAST search.

In the second report, a more elaborate method is employed to select the siRNA target sequences. This procedure exploits an observation that any accessible site in endogenous mRNA can be targeted for degradation by the synthetic oligodeoxyribonucleotide/RNase H method (5). Any accessible site identified in this fashion is then used as insert sequence in the U6 promoter-driven siRNA constructs.

Order of the sense and antisense strands within the hairpin siRNAs

A hairpin siRNA expression cassette is usually constructed to contain the sense strand of the target, followed by a short spacer, then the antisense strand of the target, in that order. One group of researchers has found that reversal of the order of sense and antisense strands within the siRNA expression constructs did not affect the gene silencing activities of the hairpin siRNA (6). In contrast, another group of researchers has found that similar reversal of order in another siRNA expression cassette caused partial reduction in the gene silencing activities of the hairpin siRNA (7). It is not clear what is responsible for this difference in observation. At the present time, it is still advisable to construct the siRNA expression cassette in the order of sense strand, short spacer, and antisense strand.

Length of the siRNA stem

There appears to be some degree of variation in the length of nucleotide sequence being used as the stem of siRNA expression cassette. Several research groups including ours have used 19 nucleotides-long sequences as the stem of siRNA expression cassette (6-10). In contrast, other research groups have used siRNA stems ranging from 21 nucleotides-long (4-5) to 25-29 nucleotides-long (11). It is found that hairpin siRNAs with these various stem lengths all function well in gene silencing studies.

Length and sequence of the loop linking sense and antisense strands of hairpin siRNA

Various research groups have reported successful gene silencing results using hairpin siRNAs with loop size ranging between 3 to 23 nucleotides (4, 6-9, 11). The following is a summary of loop size and specific loop sequences used by various research groups:

Presence of 5' overhangs in the hairpin siRNAs

Most research groups did not use a 5' overhang in their hairpin siRNA construct (4-8, 10-11). However, one research group included a 6 nucleotide 5' overhang in the hairpin siRNA constructs (9). These hairpin siRNAs with 5' overhangs were shown to be functional in gene silencing.

最初使用siRNA表达载体来诱导RNAi的研究人员对于他们编码表达siRNA的插入片段有不同的设计准则。大多数设计均具有两段反向的重复序列，之间由短间隔序列隔开，然后以一系列T作为结尾用于转录终止。这些设计得到的RNA转录本预计会折叠为如图1所示的短发卡结构siRNA。siRNA靶向序列的选择、编码发卡结构的茎（stem）既反向重复序列的长度、反向重复序列的顺序、编码发卡结构的环（loop）的间隔序列的长度及组成，以及是否带有5’端突出等设计要点，在不同报告中有着很大不同（3-11）。

图 1 .由siRNA表达载体或siRNA表达组件所得到的典型发卡siRNA及其与RNA靶向序列的关系示意图。

Ambion推荐的siRNA发卡设计程序

下文所述siRNA发卡设计及克隆方式的建议是基于Ambion的科学家的研究工作而给出的。设计合适的插入片段的第一步是根据上文“一般设计指导准则”的所述步骤来选择siRNA靶向位点。

要进行筛选，我们一般会对每个目标片段测试4条不同的siRNA序列，在基因序列的全长上将siRNA序列间隔开，以降低mRNA靶定区域高度结构化或与调控蛋白相结合的可能性。由于针对每个靶基因构建和测试四个siRNA表达载体要花费大量时间，我们发现使用基于PCR的siRNA表达组件（SEC）来筛选潜在siRNA序列要更容易。SEC是一种在发卡siRNA模板两侧带有启动子和终止子序列的PCR产物。这种筛选方法还可以鉴定实验体系中启动子和siRNA序列间的最佳组合。可以高效引起基因沉默的SEC可以快速克隆至载体中以用于长期研究。Ambion的科学家已经确定，一段序列如果作为siRNA直接转染能很好地发挥作用，在体内表达为siRNA的话同样能起到很好的效果。唯一的例外是体内表达的siRNA序列不能包含4个或5个连续的A或T，因为这会成为聚合酶III的转录终止位点。

对于传统的克隆至pSilencer载体的方法，需设计两条编码所选siRNA序列的DNA寡核苷酸以插入到载体中（图2和图3）。一般来说，这种DNA寡核苷酸需要含有19个核苷酸长的正义siRNA序列，并通过一条短间隔序列与其反向互补的反义siRNA序列相连。Ambion的科学家已成功地使用了9个核苷酸的间隔序列（TTCAAGAGA），当然也可设计其它的间隔序列。在寡核苷酸的3’末端需要添加5-6个T。另外，为了将片段克隆至pSilencer 1.0-U6载体，在DNA寡核苷酸链的5’和3’端需要分别添加EcoR I和Apa I限制性内切位点的核苷酸突出（图2）。相应的，如果要克隆到pSilencer 2.0-U6、2.1-U6、3.0-H1或 3.1-H1载体时，则需要在DNA寡核苷酸链的5’和3’端分别添加BamH I和Hind III限制性内切位点的核苷酸突出（图3）。最终获得的RNA转录本应当会发生折叠并形成茎环状结构，由19bp的主干和9nt的环以及3’端2-3个U所组成（图1）。

图 2.pSilencer 1.0-U6的插入片段设计。 该插入片段是针对pSilencer 1.0-U6载体而设计的，带有用于直接克隆至载体的相应3’端突出。环序列和长度可根据需要而变化。

图 3 .pSilencer 2.0-U6和pSilencer 3.0-H1的插入片段设计。 该插入片段是针对pSilencer 2.0-U6、2.1-U6、3.0-H1及3.1-H1表达载体而设计的，带有可直接克隆至这些质粒的相应5’端悬臂。如图2中所示的pSilencer 1.0-U6，先期研究表明环序列的设计可以有极大的随意性；这里我们提供了一条我们认为表现良好的环序列。

要将片段克隆至pSilencer腺病毒1.0-CMV载体，带茎环结构的DNA寡核苷酸可依照如上文所述的pSilencer 2.0和3.0载体中那样来设计。然而，值得注意的例外情况是，对于基于CMV的载体系统，其寡核苷酸的3’末端没有5-6个T，因为基于CMV的载体系统，其转录终止信号是SV40 polyA。另外，为了将片段克隆至pSilencer腺病毒1.0-CMV载体，在DNA寡核苷酸的5’和3’末端分别添加了带有Xho I和Spe I限制性内切位点的核苷酸悬臂（图4）。不过，对于要克隆至pSilencer 4.1-CMV载体的片段，其5’和3’末端则分别添加了带有Bam H1和Hind III限制性内切位点的核苷酸悬臂（图5）

图 4.pSilencer™腺病毒1.0-CMV载体的插入片段设计。该插入片段是针对pSilencer腺病毒1.0-CMV载体而设计的，带有用于直接克隆至载体的相应悬臂。环序列和长度可根据需要而变化。

图 5. pSilencer™腺病毒4.1-CMV载体的插入片段设计。该插入片段是针对pSilencer腺病毒4.1-CMV载体而设计的，带有用于直接克隆至载体的相应悬臂。环序列和长度可根据需要而变化。

siRNA标靶的筛选

除了我们独有的专利算法及我们建议的siRNA标靶筛选程序（即对mRNA序列进行扫描以找出AA双核苷酸，并记录下AA下游的19个核苷酸），其他研究人员还使用了两种不同的方法。在第一种方法中，siRNA靶向序列的选择是纯粹依赖经验而决定的 (4)，只要求目标序列以GG起始，且通过BLAST搜索分析与其他基因不具有明显的序列同源性。

在第二份报告中，研究人员使用了更详细的方法来选择siRNA靶向序列。该筛选程序利用了内源性mRNA的任何可接触位点均可以通过合成寡脱氧核苷酸/RNase H的方法来识别并降解这一现象 (5)。用这种方式所鉴定出的任意可接触位点可以作为插入序列用于构建U6启动子驱动的siRNA表达载体的构建。

发卡siRNA中正义链和反义链的顺序

发卡siRNA表达组件通常依次包含目标正义链、短间隔片段和目标反义链。有研究团队发现反转siRNA表达构建中的正义和反义链顺序不会影响到发卡siRNA的基因沉默活性 (6)。与此相反，另一个研究团队则发现对另一个siRNA表达组件的顺序进行类似反转会造成发卡siRNA基因沉默活性的部分降低 (7)。造成这种现象的原因尚不清楚。目前，仍建议在构建siRNA表达组件时遵循正义链、短间隔序列、反义链的排列顺序。

siRNA茎干的长度

目前看来，用作siRNA表达组件中茎干的核苷酸序列长度可以有某种程度上的变化。包括Ambion在内的几个研究团队使用19个核苷酸长度的序列作为siRNA表达组件的茎干部分 (6-10)。与此相反，其他研究团队则使用从21个核苷酸长度 (4-5) 到25-29个核苷酸长度 (11) 的siRNA茎干序列。研究发现这些具有不同茎干长度的发卡siRNA在基因沉默研究中均表现良好。

发卡siRNA中连接正义链和反义链的环的长度及序列

很多研究团队报告了使用环大小范围从3至23个核苷酸的发卡siRNA成功进行基因沉默的结果（4、6-9、11）。下表是不同研究团队所用环大小及具体序列的总结：

发卡siRNA中5’悬臂的存在

大多数研究团队在发卡siRNA构建中并未使用5’悬臂（4-8、10-11）。然而，有一个团队在发卡siRNA构建中使用了6个核苷酸长的5’悬臂 (9)。这种带有5’悬臂的发卡siRNA在基因沉默中工作正常。

Ambion synthesizes both customer designed siRNAs and siRNAs pre-designed using the Cenix algorithm.

To order a chemically synthesized siRNA for which you already have the design, you can either provide:

• the ~21 base mRNA sequence (starting with the AA dinucleotide) to which the siRNA will be directed

OR

• the sequence of each siRNA strand (This option is recommended if you wish your siRNA to have 3' termini other than dTdT or UU.)

Ambion will synthesize a complementary pair of siRNA oligonucleotides according to your sequence. By default, siRNAs for which you provide only the mRNA target sequence will be synthesized with dTdT 3' overhangs. If you wish, you can choose UU or other overhangs. Our scientists observe no functional difference in the potency of siRNA made with dTdT or UU overhangs. (Note: the 3' dTdT of the sense strand does not have to be complementary to the target gene.)

Currently, Invitrogen Pre-designed siRNAs are available for >98% of all human, mouse, and rat genes in the RefSeq database maintained by NCBI. To order a pre-designed siRNA, search our siRNA database for your gene of interest, choose the design(s) you'd like to purchase, add them to your cart, and transfer the relevant information about each to our online oligo order form. See Designing a Better siRNA for information on the design algorithm used.

Ambion可以对用户自行设计的siRNA及使用Cenix算法预设计的siRNA进行合成。

要订购您已经设计好的化学合成siRNA，您可以提供以下信息中的任意一项：

• siRNA指向的~21个碱基的mRNA序列（由AA双核苷酸起始）

或

• siRNA每条链的序列（若您希望您的siRNA带有除dTdT或UU之外的3’末端，则建议您使用该选项。）

Ambion会依照您的序列合成一对互补的siRNA寡核苷酸。若您仅提供了mRNA标靶序列，我们将默认合成带有dTdT的3’端悬臂的siRNA。若您希望，也可选择UU或其他悬臂。我们的科学家在带有dTdT或UU悬臂的siRNA的效能间没有观察到任何功能性差异。（注意：正义链的3’端dTdT无需与目标基因互补。）

目前，Ambion可对NCBI所维护的RefSeq数据库中所有人、小鼠及大鼠基因中的98%以上的基因提供 预设计siRNA 。要订购预设计siRNA，您可在我们的siRNA数据库中搜索您感兴趣的基因，选择您希望订购的设计，将它们添加至购物车中，然后将每条siRNA的相关信息填写在我们的在线oligo订购表上。关于设计所用算法的相关信息，请参见“设计更好的siRNA”。

To prepare siRNA by in vitro transcription, siRNA expression vector, or PCR-generated siRNA expression cassette, appropriate templates must be prepared. Web-based tools for designing these templates are available for the following Invitrogen kits/products:

• Silencer siRNA Construction Kit
• pSilencer siRNA Expression Vectors
• Silencer siRNA Cocktail Kit

These tools are also accessible from the siRNA Target Finder described above.

要通过体外转录、siRNA表达载体，或通过PCR得到的siRNA表达组件来制备siRNA，首先需要准备合适的模板。对于如下Ambion试剂盒/产品，可使用在线工具设计模板：

• Silencer siRNA Construction Kit
• pSilencer siRNA Expression Vectors
• Silencer siRNA Cocktail Kit

这些工具还可以通过上文所述siRNA Target Finder来获取。

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7. Paul, C.P., Good, P.D., Winer, I., and Engelke, D.R. (2002) Effective expression of small interfering RNA in human cells. Nature Biotechnology 20 : 505-508.
8. Brummelkamp, T.R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296 : 550-553.
9. Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA interference. Nature 418 : 435-438.
10. Miyagishi, M., and Taira, K. (2002) U6 promoter-driven siRNAs with four uridine 3' overhangs effectively suppress targeted gene expression in mammalian cells. Nature Biotechnology 20 : 497-500.
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• RNAi Workflow Essentials
• siRNA Design Guidelines
• Getting Started with RNAi
• Five Ways to Produce siRNAs
• siRNA Design: It's All in the Algorithm