- 产品简介
- 工作流程
- 实验数据
- siPOOLs库
- siPOOLs常见问题解答(FAQ)
- 应用文献
siPOOLs:一款特异性好的RNA干扰试剂,能够产出可靠的表型
siRNA pools(即siPOOLs)是一款经过优化设计的、高复杂性的RNAi试剂,包含了30条siRNA的混合物,经证明可有效消除脱靶效应,提高了结果的可靠性(Hannus et al., 2014)。Pack Hunter (pooling) 方法通过将单个siRNA的浓度稀释到刺激表型的阈值以下来对抗单个siRNA的脱靶效应。借助专有的设计算法,siPOOLs中的siRNA序列经过优化,以实现较大的转录本覆盖率,高效杂交,且将旁系同源基因进行了过滤,从而实现高效和特异性的基因沉默。
siPOOLs产品优势:
1. 使用简单快捷 siPOOLs与多种转染试剂兼容,几天内就能看到结果。
2. 高度特异性且有效 siPOOLs在标准细胞系中将脱靶率降低5-25倍,且在1-3 nM下实现基因敲低率≥70%。
3. 一致的表型 与siRNA相比,由siPOOL产生的表型高度一致。
4. 确保经过检验 采用RT-qPCR对siPOOL干扰效果进行验证,如果在最佳转染条件下,敲低率低于70%,则有可能重新设计。
5. 采用可信赖的基因注释进行定制设计 专业的设计,确保优化热力学特性,且避免旁系同源基因的干扰。
6. HPLC纯化且无毒 所有siPOOL均经过HPLC纯化,可降低污染物和副作用的风险。
关键问题:siRNAs的脱靶效应
科学家们将RNAi视为研究基因功能的一种快速而有效的工具。然而,短干扰RNA(siRNA)的脱靶效应和可变性能仍然是一个令人头疼的缺陷,而且在验证工作中消耗了宝贵的时间和资源。
siRNA通常与靶RNA转录本完全互补结合,通过RNAi机制指导其降解。脱靶效应主要是由siRNA模拟内源性基因调节因子microRNA(miRNA)引起的。由于miRNA只需要6个碱基种子,匹配到3'非翻译区(UTR),即可触发转录本下调。因此siRNA在通过这种机制发挥作用时,可能会改变许多意想不到的靶标基因的表达。
siPOOLs如何提高特异性?
单个siRNA或含有3-4个siRNA的低复杂度siRNA库,常常击中多个脱靶基因并表现出易变的靶基因敲低效率。
siPOOL是高度复杂且确定的30个siRNA池,每个siRNA以皮摩尔工作浓度存在。因此,siPOOL:
l 稀释了每个siRNA的脱靶效应,提高了靶向的特异性。
l 确保了靶基因的协同敲低,产生更稳健、更可靠的结果。
优化和详细的siRNA设计
在siPOOL中,可以针对特定的转录本亚型或密切相关的基因优化siRNA序列的靶向性。采用专有的siRNA设计算法,根据热力学特性选择有效的siRNA,这些特性有利于引导链加载到RNA诱导的沉默复合物(RISC)中(有关siPOOL设计的更多详细信息,请参阅技术说明)。siPOOLs使用RefSeq注释和全基因组旁系同源基因过滤,旨在以高特异性较大限度地覆盖所有靶向转录本。
使用siPOOL具有更高的特异性
特异性好的试剂,应仅影响其靶标基因。
HeLa细胞中微阵列的表达谱分析显示,单个siRNA可以诱导许多脱靶基因(红点),而针对同一靶基因(绿点)的siPOOL,可能也包含有非特异性的siRNA,但是大大降低了脱靶效应。
通过siPOOL实现更好的重现性和有效的敲低效率
siRNA的敲低效率差异很大。
与单个siRNA相比,针对同一基因的siPOOLs具有相似的敲低效率,此结果表明siPOOLs具有更高的稳健性和可重复性(左图和中图:靶RNA水平的实时定量PCR测量的相关性图),
以及siPOOLs也表现出有效的基因敲低效果。在常用的细胞系中,许多基因通常在1 nM浓度下实现75-98%的基因敲低效率(右图)。
产出您可以信赖的表型
如果RNAi效果可靠且特异性好,靶向同一基因的两种试剂理应产生相似的表型。
每个基因分别使用两个siPOOL池,一个来自我们的人类激酶siPOOL文库,一个是市售的每个基因含3条siRNAs的文库,在A549细胞中筛选了36个基因并检测了细胞活力,结果表明siPOOLs产生了较好的一致性表型。
siPOOL库使您获得值得信赖的RNAi筛选结果
——改进和节省您的RNAi筛选实验
siPOOL文库中的每个基因均被混池化的30条siRNA靶向
为什么要做RNA干扰(RNAi)?
RNA干扰(RNAi)是一种广泛用于研究基因功能的基因沉默工具。这是由于它拥有多种优势:易于使用、适用于各种细胞类型、类似药物的特性、可快速获得结果等。然而,众所周知,合成的短干扰RNA(siRNAs),通常会存在脱靶效应,这将导致实验结果的不稳定,因此后续还需要开展更多的验证工作,既费时又费力。 如果采用高复杂度的siRNAs池(简称siPOOLs),可以大大降低脱靶效应,进一步提高RNAi方法在基因功能研究中的可靠性和效率。
siPOOLs如何提高特异性?
siPOOLs是由优化设计合成的30条siRNA组成的复杂混池。采用高复杂度的混池方法,使每条siRNA的浓度均相应降低,致使特异性siRNA的脱靶效应得以稀释。相反地,siPOOLs具有较高的转录组覆盖率,可以进一步提高基因敲除的效率。由此产生的功能表型结果也更加的可靠,重复性也更好。
左:在相对较高的浓度下使用单个siRNA或低复杂度siRNA池,以实现有效的敲除。然而,高浓度可能会导致普遍的脱靶效应,从而产出易变的结果。右:siPOOLs是由30条特定的siRNAs构成的复杂混池,具有不同的种子序列,能够较大限度地覆盖靶标基因。采用混池化方法将每条siRNA的浓度降低。较低的浓度可有效消除脱靶效应,并提高基因沉默结果的可靠性。
我们提供专业的技术支持
我们的团队是由具备多年RNAi筛选经验的专家组成,在siRNA设计和RNAi筛选数据分析(包含siRNA和基于shRNA的筛选)方面有深入的专业研究,可以为您的RNAi检测开发和筛选研究提供科学、专业的技术支持。
与时俱进的siPOOL文库
为了确保siPOOLs针对当前注释的基因,设计团队会根据新版的RefSeq注释不断更新siPOOL的设计。
产品名称 | 物种 | 规格 | 产品编号 |
E3 Ligase siPOOL library E3连接酶siPOOL库 | human | 1 nmol | si-L010-000E3L |
human | 0.5 nmol | si-L005-000E3L | |
human | 0.25 nmol | si-L002-000E3L | |
human | 0.1 nmol | si-L001-000E3L | |
Kinase siPOOL library 激酶siPOOL库 | human | 1 nmol | si-L010-000505 |
human | 0.5 nmol | si-L005-000505 | |
human | 0.25 nmol | si-L002-000505 | |
human | 0.1 nmol | si-L001-000505 | |
RNA-binding protein siPOOL library RNA结合蛋白siPOOL库 | human | 1 nmol | si-L010-000RBP |
human | 0.5 nmol | si-L005-000RBP | |
human | 0.25 nmol | si-L002-000RBP | |
human | 0.1 nmol | si-L001-000RBP | |
GPCR siPOOL library GPCR siPOOL库 | human | 1 nmol | si-L010-00GPCR |
human | 0.5 nmol | si-L005-00GPCR | |
human | 0.25 nmol | si-L002-00GPCR | |
human | 0.1 nmol | si-L001-00GPCR | |
Ubiquitinase siPOOL library 泛素化酶 siPOOL库 | human | 1 nmol | si-L010-000UBI |
human | 0.5 nmol | si-L005-000UBI | |
human | 0.25 nmol | si-L002-000UBI | |
human | 0.1 nmol | si-L001-000UBI |
通常,siPOOLs产品采用携带条形码的离心管。也可根据客户需求定制96孔板或384孔板布局的siPOOLs产品。
运输和保存
siPOOL库,以悬液的形式保存,常温(RT)下运输。如有特别要求,siPOOL也可以干粉形式运输。siPOOL库可以稳定地在RT下运输至少4周。到达后,siPOOL库应存储在-20℃至-80℃。在这样的条件下,siPOOL库至少稳定保存两年。如果siPOOL库以干粉形式运输,siPOOL需采用无RNase的水进行重悬。
在客户收到(或重悬)siPOOLs产品后,我们建议您将siPOOLs分装成小体积,存储在-20℃到-80℃条件。为了达到最佳效果,尽量减少反复冻融的次数。在良好的储存条件以及规范的操作处理下,siPOOLs可稳定保存至少2年。
引用文献
Hannus M. et. al.: siPools: highly complex but accurately defined siRNA pools eliminate off-target effects. Nucleic Acids Res 42(12): 8049-61(2014)
Marine S. et. al.: Common Seed Analysis to Identify Off-Target Effects in siRNA Screens. Journal of Biomolecular Screening 1-9 (2011)
Jackson A. et. al.: Expression Profiling reveals off-target gene regulation by RNAi. Nature Biotechnology (2003)
1. siPOOLs如何提高基因敲低的特异性和效率?
siPOOL是一款包含30条siRNAs的、高复杂度的混池,通过降低每条siRNA的实验相关性以稀释其脱靶效应。专有的siPOOL设计算法,可确保较高的转录本覆盖率,且避免旁系同源基因,提高基因敲低的效率和特异性。此外,生产的siPOOL包含了规定长度和高纯度的siRNA标准品,从而大大降低非特异性影响的风险。
2. siPOOLs与其他市售siRNA池有什么区别?
其他市售siRNA池要么是3-4个siRNA的低复杂度混池,要么是不同siRNA长度的随机混池。相比之下,siPOOLs是一款好用、高复杂度、确定长度的siRNA混池。
3. 如果我订购一个siPOOL,总量有多少?我可以用它做多少个反应?
对于每个靶基因,siPOOL的规格有5、10或20 nmol。当siPOOL浓度为1nM时,5 nmol至少足够用于6孔板的2250个孔或者96孔板的45000个孔(参见siPOOL转染方案)。10个及以上的siPOOL批量订单,可以定制1-2 nmol的小规格。请联系我们获取定制报价。
4. 从下单到交货需要多长时间?
我们需要大约3-4周的时间来交付新的siPOOL。而对于已有库存的siPOOLs,可在2周内交货。根据序列的不同,某些siPOOLs可能需要更长的时间来合成。我们将会根据实际情况及时和您沟通是否会延迟交货。
5. siPOOL可以针对的最小序列长度是多少?
通常,目标基因的特有序列≥300个碱基就可以设计siPOOL。在保持种子序列多样性的条件下,siRNA之间存在一定程度的序列重叠是没有问题的。请将您的序列信息发送给我们,经过评估后我们会告知您设计的可行性及建议。
6. 购买siPOOL有什么保证呢?
针对所有编码基因的siPOOLs均在包含验证的保证范围内。当以高达10nM浓度使用siPOOL,且在转染24小时后做RNA定量检测分析,我们保证敲低率≥70%。在表达给定靶标的标准细胞系中,阳性对照siRNA应获得最佳的转染条件。如果没有看到≥70%的敲低率,我们将根据可用的序列免费为客户重新设计、合成、验证和发货一个新的siPOOL。由于敲低效率也会随着基因的特性而发生变化,因此使用新的siPOOL能否提高敲低效率,我们无法做出保证。
针对非编码基因的siPOOL,如果未达到≥60%的敲低率,则根据可用的序列进行重新合成。不过,验证和运输的费用需由客户自己承担。
7. 为什么不提供针对长非编码RNA(lncRNA)的siPOOL验证?
由于二级结构、结合蛋白或细胞定位等因素限制了RNAi机制发挥作用,因此通过RNAi沉默长非编码RNA(lncRNA)更具挑战性。根据可用的转录序列,我们将提供一轮siPOOL重新设计和合成,以靶向lncRNA的不同区域。然而,siPOOL的验证工作最好还是由熟悉该lncRNA特性的客户自己开展。
8. 可以提供免费样品进行测试吗?
目前,只有0.1 nmol阳性对照(GAPDH / KIF11 / INCENP)和阴性对照siPOOLs可供免费测试。我们欢迎您对经常评估的基因提出建议,未来将有可能会将其作为阳性对照。
9. 能否以更低的价格提供较小规格的siPOOL给到我们进行测试或筛选?
≥ 10个 siPOOLs的批量订单,可以提供较小的规格,如1-2 nmol。人激酶siPOOL库和siPOOL癌症工具箱中的预定义siPOOLs也可用于制定较小的规格。请联系我们获取报价。
10. 对于核定位的RNA,siPOOLs有效吗?
已经有存在于细胞核中的RNAi机制的相关报道,一些针对核定位RNA(例如MALAT1,XIST)的siPOOLs可以达到70%的敲低效率。然而,细胞质定位的RNA可能会更有效地被RNAi靶向。
11. siPOOLs可以组合在一起同时敲低几个基因吗?
是的。siPOOLs在低纳摩尔浓度下是有效的。这允许在单个应用中组合多个siPOOLs,以沉默多个基因,同时将副作用的风险降至最低。
在一项研究中,siPOOLs同时成功地沉默多达四个基因的表达 [Welsbie, D. S. et al. Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons. Neuron 94(6), 1142–1154.e6 (2017)]。
12. 我应该使用什么浓度的siPOOL?
在标准细胞系(例如HeLa,Hek293,A549,MCF7)中,siPOOL通常在借助脂质体转染试剂的情况下采用1-3 nM的浓度。在难转染的细胞中,可能需要更高的浓度,并且可以采用电穿孔等替代方法。我们建议先设置不同的浓度梯度,以确定具有稳定敲低效率的最低浓度。对于长期的基因敲低(>3天),建议使用更高的初始siPOOL浓度。另外,我们也可以为您提供剂量优化服务,我们会采用7个浓度梯度进行试验。如需了解更多信息,请联系我们。
13. siPOOL介导的基因敲低效果能持续多久?
siPOOL敲低基因的持续时间与其他siRNA相似,取决于细胞系和靶基因。基因敲低效果通常可持续4-7天。但是,高增殖率的细胞或高活跃度的基因可能会维持更短的时间。通过siPOOLs的再转染或提高siPOOL初始浓度的转染,也许可以延长基因敲低效果的持续时间。
14. siPOOL可以靶向特定的亚型吗?
已证明,siPOOL能够以高特异性和高效率成功地靶向选定亚型。然而,成功概率还取决于该亚型特有序列的可用性。如有需要,请将您的转录本NCBI ID发送给我们,我们可以为您作出评估。另外,我们还可为您提供跨亚型或物种选择性的siPOOL验证服务。
15. siPOOL的阴性对照是什么?
我们使用不与人类、小鼠和大鼠基因相互作用的标准阴性对照siPOOL(30个siRNAs)。它已在多个细胞系中进行了测试,对细胞增殖、凋亡或细胞形态没有显著影响。另外,我们也可以根据需要提供Scrambled阴性对照siPOOL,对靶siRNA序列进行加扰以避免靶标相互作用,同时保持GC含量的百分比(%)。
16. 您的阳性对照siPOOL是什么,读数是多少?
阳性对照siPOOL是预先经过验证的siPOOL,当转染细胞时会产生确定的表征,表明转染成功。目前,可用的阳性对照siPOOL所对应的基因,包括:人KIF11(3832),可产生有丝分裂停滞和可观察到的细胞死亡现象;人INCENP (3619),可产生在显微镜下可观察到的细胞增大表型;人GAPDH(2597),其不产生可观察到的表型,但通过定量PCR检测GAPDH的RNA水平时,表现为显著的下调。也可提供针对小鼠的GAPDH(14433)和KIF11(16551)的阳性对照siPOOL。
17. siPOOLs可以针对除人类,小鼠和大鼠以外的其他物种吗?
是的,siPOOLs可以针对任何物种。请向我们提供目标物种、宿主系统和目标序列。
18. siPOOLs的转染条件与其他siRNA有什么不同吗?
没有太大的差别。您可以将已经优化好的siRNA转染条件应用于siPOOLs转染中。
19. 有推荐的转染试剂吗?
市面上的转染试剂种类繁多。对于许多常见的细胞系,采用Lipofectamine的转染效果就很好。然而,原代巨噬细胞或非贴壁细胞等细胞类型的转染可能会存在更多的挑战。我们可为您提供细胞系转染优化服务,我们将测试3种转染方法。如需了解详情,请联系我们。
20. siPOOL的保质期是多久?
siPOOLs在-20°C下储存时,至少可以稳定6个月。尽管我们观察到siPOOL在保存几年后仍存在活性,但是,我们推荐您在收到产品后尽快使用,尽量不超过保质期。另外,强烈建议您将较大的体积分装成小体积再进行保存,以避免多次反复冻融影响实验结果。
21. siPOOL可以作为一种可发表的验证方法吗?
是的。siPOOLs不仅可以用于常规的基因敲低实验,而且可以作为其他技术(如CRISPR,单个siRNA或shRNA)的补充验证方法。在使用siPOOLs进行发表时,请引用“siTOOLs Biotech”以及Hannus et al., Nucleic Acids Res, 2014文献。
22. 是否可以进一步的验证siPOOL,例如:开展回补实验?
是的。siPOOL敲低效果的进一步验证,可以采用抗siPOOL回补序列(siPOOL-resistant rescue constructs)来开展,它可以表达siPOOL抗性版本的基因以挽救和恢复目标基因的功能。
23. 是否需要反卷积一个siPOOL,来单独测试siRNAs?
我们不建议对siPOOL进行反卷积,因为这会破坏高复杂性混池化所赋予的高特异性。为了进一步验证siPOOL结果,我们建议使用抗siPOOL回补序列。
24. siPOOL以什么形式发货?它们在室温下稳定吗?
siPOOL以悬浮液的形式发货(10 nM Tris,pH 8.0)。siPOOL经测试可在室温(RT)下稳定至少4周,在37°C下稳定1周,在50°C下稳定24小时。因此,温暖气候条件下的运输延迟,不太对siPOOL的活性产生不利的影响。
25. siPOOL文库是否可用于功能基因组筛选?
是的。我们有一个包含505个siPOOLs的siPOOL人类激酶库,可用于高通量功能基因组筛选,以获得可靠的RNAi效果。另外,用于RNA结合蛋白、E3连接酶和去泛素酶的siPOOL文库,以及其他定制化文库,请联系我们咨询。
siPOOLs 产品已协助许多的学术研究人员、科学家、制药/生物技术公司和RNAi筛选人员将论文发表在《自然》、《细胞》、《自然医学》、《科学报告》等顶级期刊上。期待与您的合作,我们将为您的RNA研究提供量身定制的分子工具。
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