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查看更多产品信息 Ambion™ mirVana™ miRNA Inhibitor, let-7c positive control - FAQs (4464081, 4464080)
31 个常见问题解答
使用TaqMan miRNA Assay，您可以测定感兴趣的miRNA的水平。如果miRNA与转染进细胞的anti-miR和/或mirVana抑制物相结合，那么miRNA将不会被检测到，证明miRNA的有效失活。请注意，这仅适用于当提取的总RNA样本在加入RT引物时没有进行热变性的情况，因为热变性将导致复合体解体，这样即使细胞已被正确的转染，TaqMan miRNA Assay将仍能检测到miRNA。更多细节请参考TaqMan MicroRNA Cells-to-CT试剂盒（货号4391848）的实验方案。
模拟物和抑制物可以使用和siRNA相同的参数通过转染或电转进入细胞。
使用mirVana miRNA模拟物
为了后续应用，请使用一种对于您感兴趣的miRNA表达相对较低的细胞系。
• 转染时的细胞密度通常建议30–70%汇合度。
• 转染方法（传统转染或反式转染）。
• 细胞暴露于转染试剂-RNA寡核苷酸复合体的时长。
对于大部分miRNA模拟物而言，最大活性在转染后24小时候产生，但是生物检测实验可以在转染后24–72小时内进行。如果转染后的细胞培养超过24小时，我们建议更换新鲜的培液以便获得更好的细胞活力。
使用 mirVana miRNA抑制物分析内源miRNA功能
为了后续应用，请使用一种对于您感兴趣的miRNA表达相对较高的细胞系。
通用转染起始点
mirVana miRNA模拟物和抑制物通常在3–30 nM浓度转染时效果最好。优化实验可以包括1–100 nM的浓度范围。在准备转染复合体时，为提高精确性和可重复性，可以先稀释您的寡聚核苷酸储液，并加入较大体积的稀释后的溶液。
交货时间取决于纯化程度和是否是定制订单。请参考以下大致时间表（中国需要加上国际运输时间）：
标准纯化(5 and 20 nmol) ：4个工作日
HPLC/in vivo ready (250 nmol)：15个工作日
定制mirVana抑制物和模拟物：15个工作日
是的，请将您未发表的miRNA序列提供给我们，我们可以合成一个相应的mirVana模拟物或抑制物，请点击此链接（https://www.thermofisher.com/order/custom-genomic-products/tools/mirna/）订购。
阴性对照的提供需要签署机密信息披露协议（confidential disclosure agreement），而目标序列的提供需要提供购买证明。
将干粉状的寡聚核苷酸保存在–20°C或更低温度下的非自动除霜的冰箱中。将重悬后的寡聚核苷酸保存在–20°C或更低温度下，可保存较长时间（可长至一年），并且它们可经受多至50次的反复冻融而不会产生显著降解。我们建议将重悬后的miRNA分装以避免污染。可以存储在–80°C但并不必要。工作溶液可以在使用前保存在4°C多至7天。
是的，我们对于mirVana miRNA抑制物提供阳性和阴性对照。mirVana miRNA抑制物let-7c阳性对照被用来在哺乳动物细胞内进行mirVana miRNA抑制物(货号4464080, 4464081)实验时的阳性对照。内源let-7c miRNA被证明可以负向调控 HMGA2 mRNA在培养细胞中的表达。HMGA2是一个广泛表达的非组蛋白的染色质蛋白，可通过改变染色质构象而调控基因表达。let-7c miRNA可以敲低HMGA2 mRNA水平。当转染进人和小鼠细胞系内时，let-7c miRNA抑制物会抑制内源let-7c miRNA，导致HMGA2 mRNA水平提高。因此，可以通过实时RT-PCR检测HMGA2 mRNA而对let-7c miRNA抑制物在人和小鼠细胞内的活性进行监控。
mirVana miRNA抑制物阴性对照#1在进行mirVana miRNA抑制物(货号4464076, 4464077, 4464078, 4464079)实验时用作阴性对照。将此阴性对照和阳性对照（如let-7c阳性对照）和实验组mirVana miRNA抑制物作为平行实验同时转染。使用阴性对照转染样本中目的基因的表达作为基线评估对照和实验组miRNA抑制物对目的基因的表达调控。
是的，我们提供mirVana miRNA模拟物的阳性和阴性对照。mirVana miRNA Mimic miR-1阳性对照（货号4464062, 4464063, 4464064, 4464065）被设计用于在哺乳动物细胞内模拟成熟miR-1 miRNA。miR-1和很多基因的敲低相关。特别是miR-1介导的Protein Tyrosine Kinase 9 (PTK9)敲低已被证明发生在mRNA水平。miR-1阳性对照可以作为mirVana miRNA模拟物进行转染时的阳性对照。使用实时RT-PCR检测PTK9 mRNA的表达来监控miR-1阳性对照的活性。
mirVana miRNA模拟物的阴性对照 #1 (4464058, 4464059, 4464060, 4464061)设计作为使用mirVana miRNA模拟物时的阴性实验对照。转染阴性对照的方法与阳性对照 (如miR-1 Positive Control)和实验组mirVana miRNA模拟物的方法相同。使用转染阴性对照的样本中目的基因的表达作为本底来评估对照和实验组miRNA模拟物对目的基因的表达调控。mirVana miRNA模拟物阴性对照 #1是一个随机序列的miRNA模拟物，它已经在人类细胞系和组织中被广泛验证不会对已知miRNA产生可检测的效应。
mirVana miRNA模拟物的平均分子量是14,000 g/mol (1 nmol = 14 µg)，mirVana miRNA抑制物的平均分子量是11,000 – 12,000 g/mol (1 nmol = 11 – 12 µg)。精确的分子量是保密的。
请访问miRBase（http://www.mirbase.org/）: The microRNA database（microRNA数据库）。
我们可以对Pre-miR和Anti-miR miRNA进行标记，但不提供标记的mirVana模拟物和抑制物。5’修饰包括Alexa Fluor 555, Alexa Fluor 647, FAM, 和Cy3。请点击此处（https://www.thermofisher.com/us/en/home/global/forms/custom-sirna-mirna-quote-request.html）询价。
是的，请提交您的未发表的miRNA序列并/或点击此处（https://www.thermofisher.com/order/custom-genomic-products/tools/mirna/）订购定制修饰的miRNA。
Ambion Anti-miR抑制物和Ambion Pre-miR miRNA模拟物是我们的第一代抑制物和模拟物。Ambion Anti-miR抑制物是化学修饰的单链核酸，设计用于特异性结合并抑制内源microRNA (miRNA)分子。它们是可以使用转染或电转导入到细胞内的抑制物。针对miRBase version 15.0中的miRNA我们均设计了抑制物。
Ambion Pre-miR miRNA模拟物是化学修饰的短双链RNA分子，设计用于模拟内源成熟的miRNA。这些miRNA模拟物类似于siRNA，但不等同于siRNA，它们可以使用类似于siRNA的参数被转染或电转导入到细胞内。请注意，Pre-miR miRNA模拟物虽然叫Ambion Pre-miR miRNA Precursors，但并不是发夹结构，不能和miRNA前体相混淆。
mirVana miRNA模拟物和抑制物是我们的第二代模拟物和抑制物。mirVana miRNA模拟物是化学修饰的小双链RNA，它模拟内源成熟miRNA并通过上调miRNA活性使得研究miRNA功能成为可能。相比第一代模拟物它们的特异性有所提高，并且经HPLC纯化，可直接用于转染。
miRNA抑制物是化学修饰的小单链RNA分子，设计用于特异性结合并抑制内源miRNA分子并通过调低miRNA活性使得研究miRNA功能成为可能。相比第一代抑制物它们的性能有所提高。我们针对miRBase version 19.0中的miRNA均设计了抑制物。它们使用HPLC纯化，并可直接用于转染。想了解更多关于mirVana miRNA模拟物和抑制物的信息，请点击此视频（https://www.youtube.com/watch?feature=player_embedded&v=Q3MO1lmT0kw）。模拟物和抑制物均可以单独或作为文库订购。
它们既可用于体外也可用于体内实验，并且已验证可与Lipofectamine RNAiMAX转染试剂连用用于细胞水平的实验，和Invivofectamine 3.0试剂连用用于体内实验。这些寡核苷酸无毒并且在所测试的动物模型中未引发免疫反应。In vivo–ready mirVana miRNA模拟物和抑制物使用HPLC纯化并经过透析，可以直接用于体内实验。对于所有文库咨询，请电邮：rnailibraries@lifetech.com。
siRNA通过序列特异的方式介导目标mRNA分子的降解，而miRNA抑制翻译而不剪切mRNA。
miRNA (microRNA)是植物和动物基因组中天然产生的短链、高度保守的小非编码RNA分子。miRNA是单链的，长18-25nt并且可以调控转录后mRNA水平，它们通常结合互补mRNA序列的3’非翻译区(3’-UTR)，导致翻译抑制和基因沉默。研究表明数以千计的人类蛋白编码基因被miRNA所调控，说明miRNA是很多重要生物进程的“重要调控者”。
Using TaqMan miRNA assays, you can measure the level of miRNA of interest. If the miRNA is bound to the synthetic anti-miRs and/or mirVana inhibitors transfected, the duplex should not be detected, indicating efficient miRNA inactivation. Please note, this only works when total RNA sample isolated is not heat denatured when RT primer is added, as heat denaturation will result in complex dissociation and subsequent detection of microRNA by TaqMan miRNA assay even when cells have been transfected properly. Please see the protocol for the TaqMan MicroRNA Cells-to-CT kit (Cat. No. 4391848) for more details.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Mimics and inhibitors can be transfected or electroporated into cells using parameters similar to those used for siRNA.
Using mirVana miRNA Mimics- For the following applications, use a cell line that expresses relatively low levels of the mature miRNA of interest.
- Cell density at the time of transfection. In general, 30-70% confluency is recommended.
- Transfection method (traditional or reverse transfection).
- Length of exposure of cells to transfection agent-RNA oligonucleotides complexes.
For many miRNA mimics, maximal activity is achieved after 24 hours, but biological assays may be performed 24-72 hours posttransfection. If you maintain your transfected cells longer than 24 hours, we suggest replacing the existing medium with fresh medium at 24 hours posttransfection, for greater viability of the cells.
Using mirVana miRNA Inhibitors to analyze endogenous miRNA function
For the following applications, use a cell line that expresses relatively high levels of the mature miRNA of interest.
General transfection starting points
mirVana miRNA Mimics and Inhibitors typically work best when transfected at a final concentration of 3-30 nM. Optimization experiments might include a concentration range of 1-100 nM. To increase accuracy and reproducibility when preparing transfection complexes, prepare a dilution of your stock oligonucleotides, and pipet a higher volume of diluted stock.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
The turnaround time varies depending on the level of purification and whether it is a custom order or not. Please see the approximate timetable below:
Standard purification (5 and 20 nmol), 4 business days
HPLC/in vivo ready (250 nmol), 15 business days
Custom mirVana Mimics and Inhibitors, 15 business days
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Yes, please provide us with your unpublished miRNA sequence and we can synthesize a corresponding mirVana mimic or inhibitor by clicking on this link (www5.invitrogen.com/custom-genomic-products/tools/mirna/).
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Negative control sequences require a confidential disclosure agreement while target sequences require prior proof of purchase for release.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Store the dried oligonucleotides in a non-frost-free freezer at or below -20 degrees C. Store resuspended oligonucleotides at or below -20 degrees C for extended periods (up to 1 year), and they can undergo up to 50 freeze/thaw cycles without significant degradation. We recommend aliquoting the resuspended miRNA to prevent contamination. Storage at -80 degrees C is fine but not necessary. Working stock solutions can be stored at 4 degrees C for up to 7 days before use.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Yes, we offer both positive and negative controls for our mirVana miRNA inhibitors. The mirVana miRNA Inhibitor let-7c Positive Control is intended for use as a positive control for mammalian cell culture experiments using mirVana miRNA Inhibitors (Cat. No. 4464080, 4464081). Endogenous let-7c miRNA has been shown to negatively regulate HMGA2 mRNA in cultured cells. HMGA2 is a ubiquitously expressed, non-histone, chromatin protein that can modulate gene expression through changes in chromatin architecture. let-7c miRNA down-regulates levels of HMGA2 mRNA. When transfected into human and mouse cell lines, let-7c miRNA inhibitor blocks endogenous let-7c miRNA, resulting in increased levels of HMGA2 mRNA. Thus, let-7c miRNA inhibitor activity can be monitored in human or mouse cells using real-time reverse transcription PCR (RT-PCR) to detect HMGA2 mRNA.
mirVana miRNA Inhibitor Negative Control #1 is intended for use as a negative control for experiments using mirVana miRNA Inhibitors (Cat. No. 4464076, 4464077, 4464078, 4464079). Transfect this negative control in parallel with your positive control (such as let-7c Positive Control) and experimental mirVana miRNA Inhibitors. Use target expression from negative control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA inhibitor on target gene expression.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Yes, we offer both positive and negative controls for our mirVana miRNA mimics. The mirVana miRNA Mimic miR-1 Positive Control (Cat. No. 4464062, 4462063, 4462064, 2262065) is designed to mimic mature miR-1 miRNA after delivery to mammalian cells. miR-1 is associated with down-regulation of many genes. In particular, miR-1-mediated down-regulation of Protein Tyrosine Kinase 9 (PTK9) has been shown to occur at the mRNA level. miR-1 Positive Control is intended for use as a positive control for transfection experiments using mirVana miRNA Mimics. Monitor activity of miR-1 Positive Control through its gene silencing of PTK9, using real-time reverse transcription-PCR to detect PTK9 mRNA.
mirVana miRNA Mimic Negative Control #1 (4464058, 4464059, 4464060, 4464061) is designed to use as a negative control for experiments using mirVana miRNA Mimics. Transfect the Negative Control using the same methodology as for your positive control (such as miR-1 Positive Control) and experimental mirVana miRNA Mimics. Use target gene expression from Negative Control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA mimic on target gene expression. mirVana miRNA Mimic Negative Control #1 is a random sequence miRNA mimic molecule that has been extensively tested in human cell lines and tissues and validated to not produce identifiable effects on known miRNA function.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
The average molecular weight for mirVana miRNA mimics is 14,000 g/mol (where 1 nmol = 14 µg), and 11,000 - 12,000 g/mol (where 1 nmol = 11 - 12 µg) for mirVana miRNA inhibitors. The exact molecular weights are proprietary.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Please visit miRBase: The microRNA database (http://www.mirbase.org/).
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We can label our Pre-miR and Anti-miR miRNA, but do not offer labeled mirVana Mimics and inhibitors. 5' modifications include Alexa Fluor 555, Alexa Fluor 647, FAM, and Cy3. Request a quote here (http://www.thermofisher.com/us/en/home/global/forms/custom-sirna-mirna-quote-request.html).
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Yes, submit your unpublished miRNA sequences and/or order custom modified miRNA here (http://www.thermofisher.com/order/custom-genomic-products/tools/mirna/).
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Invitrogen Anti-miR inhibitors & Invitrogen Pre-miR miRNA Precursors are our first generation mimic and inhibitors. Invitrogen Anti-miR miRNA Inhibitors are chemically modified, single stranded nucleic acids designed to specifically bind and inhibit endogenous microRNA (miRNA) molecules. These are ready to use inhibitors that can be introduced into cells using transfection or electroporation. Pre-designed inhibitors have been created for all miRNAs listed in miRBase version 15.0.
Invitrogen Pre-miR miRNA Precursors are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. These ready-to-use miRNA mimics, which are similar to, but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs. Please note, Pre-miR miRNA Precursors are not hairpin constructs and should not be confused with pre-miRNAs.
mirVana miRNA mimics and inhibitors are our second generation of mimics and inhibitors. mirVana miRNA mimics are small, chemically double-stranded RNAs that mimic endogenous mature miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity. They have enhanced specificity over the first generation mimics, are delivered as HPLC purified, and ready to transfect.
miRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down-regulation of miRNA activity. They have enhanced potency over our first generation inhibitors. Pre-designed inhibitors were created for all miRNAs listed in miRBase version 19.0. They come delivered as HPLC purified, and ready to transfect. Learn more about mirVana miRNA mimics and inhibitors through this video (https://www.youtube.com/watch?feature=player_embedded&v=Q3MO1lmT0kw). Both mimics and inhibitors are available individually or as libraries.
They are compatible with both in vitro and in vivo applications, and have been validated with Lipofectamine RNAiMAX Transfection Reagent for use in cell-based systems, and with Invivofectamine 3.0 Transfection Reagent for in vivo delivery. These oligonucleotides are nontoxic and do not induce an immune response in the animal models tested. In vivo-ready mirVana miRNA mimics and inhibitors have been purified by HPLC and dialysis and are ready for immediate in vivo use. For all library inquiries, please email: rnailibraries@lifetech.com.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
siRNA mediates degradation of a target mRNA molecule in a sequence-specific manner, while miRNA suppresses translation without cleaving mRNA.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
miRNA (microRNAs) are short, highly conserved small noncoding RNA molecules naturally occurring in the genomes of plants and animals. miRNAs are single stranded, 18-25 nucleotides long and regulate posttranscriptional mRNA expression, typically by binding to the 3' untranslated region (3'-UTR) of the complementary mRNA sequence, resulting in translational repression and gene silencing. Studies have shown that thousands of human protein-coding genes are regulated by miRNAs, indicating that miRNAs are “master regulators” of many important biological processes.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
These products are delivered dry lyophilized.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.