AllStars Negative Control siRNA

RNAi实验的阴性对照

  • 经最全面验证的阴性对照
  • 与已知的哺乳动物基因无同源性
  • 最小的非特异性效果
  • 可用于miRNA模拟实验

AllStars Negative Control siRNA是目前经过最全面的测试和验证的阴性对照siRNA。该siRNA与已知的哺乳动物基因无同源性。已经运用Affymetrix GeneChip芯片和多种细胞实验验证了其有效性,确保该siRNA对基因表达和表型的非特异性效应最小。通过比较基因特异性siRNA和阴性对照,可以得出靶基因敲减对基因表达和表型的影响,而最小的非特异性效应确保这一结论真实可靠。如果阴性对照引起非特异性效应,则RNAi实验结果会被干扰且难以解释。克隆实验证实AllStars Negative Control siRNA进入RISC。AllStars Negative Control siRNA正处于专利申请中,该序列是专有的。

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Thoroughly tested and validated nonsilencing siRNA with no homology to any known mammalian gene,
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The AllStars Negative Control siRNA is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Low nonspecific effects on expression.|Cell number unaffected.|AllStars Negative Control siRNA is incorporated into RISC.|Western analysis shows AllStars Negative Control siRNA enters RISC.|Normal DNA synthesis phenotype.|No increase in cytotoxic effects.|Normal cell-cycle distribution.|Reporter construct for RISC-incorporation experiment|Nuclear size phenotype unaffected.|
Multiple negative control siRNAs (Control 1– Control 10) were transfected in triplicate into MCF-7 cells. After incubation, cRNA was prepared and hybridized to Affymetrix human U133 GeneChip arrays. Regulated genes were identified as genes that showed at least a 1.5-fold change in expression (both upregulated and downregulated) compared to untransfected cells. Ingenuity pathway analysis software was used to determine the proportion of regulated genes in each pathway compared to the total number of genes identified as central to that pathway. Where a bar appears in the figure, this means that genes in the pathway were regulated by the siRNA. If every pathway gene was regulated, the relative proportion would be 100%. Lower bars therefore indicate a lower relative proportion of regulated genes within that pathway. Where no bar appears, no genes of the pathway were regulated by the siRNA. AllStars Negative Control siRNA (indicated with arrow) resulted in the lowest number of regulated genes. In contrast, other control siRNAs resulted in higher numbers of regulated genes from important cellular pathways.|Identical numbers of MCF-7 cells were transfected with AllStars Negative Control siRNA or another negative control siRNA (Control 1). Untransfected cells were also analyzed. After 72 hours, cells were harvested, washed in PBS, transferred to TruCOUNT tubes, and counted using FACS analysis.|

MCF-7 and HeLa cells were cotransfected with the reporter construct and either a noncomplementary siRNA or AllStars Negative Control siRNA. After 24 hours, expression of the fluorescent reporter gene was measured by [A] fluorescence microscopy (HeLa cells shown) and [B], [C] FACS analysis. Normal fluorescence was observed after cotransfection with the noncomplemetary siRNA, showing that the reporter gene is expressed. After cotransfection with AllStars Negative Control siRNA fluorescence was significantly decreased, showing that the reporter gene is downregulated.

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HeLa cells were cotransfected with the reporter construct and either a noncomplementary siRNA or AllStars Negative Control siRNA. After 48 hours, western blot analysis was used for measurement of expression of the His tag. The His tag was expressed after cotransfection with the noncomplementary siRNA. After cotransfection with AllStars Negative Control siRNA, the His tag was not detected.

|HCT-116 cells (6 x 104) were [A] untransfected or transfected with [B] AllStars Negative Control siRNA or [C] another negative control siRNA (Control 1). After 72 hours, cells were treated with BrdU, fixed, and permeabilized. Cells were then stained using anti-BrdU–FITC antibody. DNA synthesis rates were measured by determining the percentage of BrdU-positive cells (region M2) using FACS analysis. The percentage of BrdU-positive cells was similar in untransfected cells and cells transfected with AllStars Negative Control siRNA (53.1% and 52.5% respectively). In contrast, cells transfected with Control 1 showed only 10% BrdU-positive cells indicating altered DNA synthesis.|Identical numbers of MCF-7 cells were [A] untransfected or [B] transfected with AllStars Negative Control siRNA, or [C] transfected with another negative control siRNA (Control 7). After 72 hours, all cells from the culture flasks were stained with propidium iodide, counted by FACS, and the number of living (propidium iodide negative) and dead (propidium iodide positive) cells were determined. Transfection of AllStars Negative Control siRNA resulted in similar numbers of dead cells as seen for untransfected cells (10.8% and 10.1% dead cells, respectively). The other control siRNA caused an increase in the level of cell death to 28.7%.|MCF-7 cells were [A] untransfected or [B] transfected with AllStars Negative Control siRNA or [C] CDC2 siRNA. CDC2 siRNA was transfected as a positive control, as knockdown of CDC2 is known to affect the cell cycle and result in accumulation of cells in the G2 phase. After 72 hours, cells were detached from the culture plate using trypsin and fixed in 70% ethanol prior to treatment with RNase and propidium iodide staining. FACS analysis was performed for 40,000 cells of each sample. Cell-cycle distribution for cells transfected with AllStars Negative Control siRNA was similar to that observed for untransfected cells. Cell-cycle distribution for cells transfected with CDC2 siRNA showed accumulation of cells in the G2 phase as expected.|

The reporter construct consisted of artificial target sequence complementary to AllStars Negative Control siRNA fused to a fluorescent reporter gene with a His tag.

|HCT 116 cells were transfected with AllStars Negative Control siRNA, another negative control siRNA (Control 1), and CDC2 siRNA. CDC2 siRNA was transfected as a positive control, as knockdown of CDC2 is known to affect the cell cycle and result in enlarged nuclei. Untransfected cells were also analyzed. After 72 hours, the nuclei of live cells were stained with Hoechst 33342 and the surface area of individual cell nuclei was measured using the cell^R Imaging System (Olympus). In this graph, 100 nuclear size measurements per treatment were plotted in order of size. AllStars Negative Control siRNA resulted in a nuclear size profile similar to untransfected cells. CDC2 siRNA resulted in enlarged nuclei as expected. The negative control siRNA, Control 1, also resulted in enlarged nuclei.|
Performance

表中实验用来确证AllStars Negative Control siRNA的性能特征。

进行的实验
实验类型 实验名称 目的 使用AllStars Negative Control siRNA的结果
全基因分析 Affymetrix GeneChip芯片 非特异性调控基因表达 最少数目的基因被调控
细胞水平分析 活细胞核染色 核大小 正常
细胞水平分析 细胞数目 增殖速率 无变化
细胞水平分析 核酸整合 DNA合成率 无变化
细胞水平分析 活细胞染色排除 细胞毒性作用 无变化
细胞水平分析 DNA染色 细胞周期分布 正常
RISC整合分析(HeLa和MCF-7细胞) 报告质粒转染 确定siRNA是否整合入RISC(一个有效的阴性对照应整合入RISC) 整合入RISC
Affymetrix GeneChip芯片

转染多种阴性对照siRNA后,运用全基因组分析检测它们对基因表达的非特异性效应。使用HiPerFect Transfection Reagent将不同来源的多种阴性对照siRNA转染至MCF-7、K562和原代HUVEC细胞。这些阴性对照siRNA包括非沉默siRNAs(与哺乳动物基因无同源性)、干扰siRNAs(碱基组成与基因特异性siRNA相同而碱基序列不同的siRNAs)和靶向人工合成报告基因的siRNAs。随后,用Affymetrix GeneChip芯片测试人全基因谱的表达。使用AllStars Negative Control siRNA,非特异性调控基因最少,适合做阴性对照。而其它的阴性对照siRNAs则导致许多源于细胞通路的基因的非特异性调控(参见"Low nonspecific effects on expression")。

活细胞染色

活细胞核染色用于测量细胞核的大小。细胞核大小的变化可作为细胞周期紊乱或生长抑制的信号。我们测试了一系列不同类型的阴性对照siRNAs,AllStars Negative Control siRNA结果最好。与未转染的细胞相比,转染AllStars Negative Control siRNA不会改变细胞核的大小。相反,转染其他的阴性对照siRNAs会使细胞核增大(参见"Nuclear size phenotype unaffected")。

细胞数目

转染一系列阴性对照siRNAs后,进行细胞计数以确定细胞增殖是否正常。与未转染细胞相比,转染AllStars Negative Control siRNA后细胞数目几乎无差异。相反,转染其他的siRNAs后细胞数目显著下降,如Control 1,表明这些siRNAs会导致生长缺陷表型(参见"Cell number unaffected")。

核酸整合

通过检测未转染和转染了一系列不同阴性对照siRNAs的HCT-116细胞的核酸整合,以确定DNA合成率。通过检测溴脱氧尿嘧啶核苷(BrdU)的摄取来测量核酸整合,BrdU是一种在DNA复制过程中代替胸腺嘧啶并可整合进新合成的DNA的胸腺嘧啶碱基类似物。DNA合成率的改变预示着细胞生长和细胞周期的变化。使用AllStars Negative Control siRNA转染的细胞,BrdU整合率与未转染细胞非常相似。然而,使用其他阴性对照siRNAs转染细胞,DNA合成率则较低,这表明该siRNA影响了细胞生长和细胞周期(参见"Normal DNA synthesis phenotype")。

活细胞染色排除

使用活细胞染色排除实验测一系列阴性对照siRNAs潜在的细胞毒性。结果显示,使用AllStars Negative Control siRNA转染的细胞与未转染细胞具有相同的活细胞数目。相反,其他的阴性对照siRNAs则导致细胞毒性增加(参见"No increase in cytotoxic effects")。

DNA染色用于细胞周期分析

细胞固定后用DNA染色法检测细胞周期的分布(处于G1/G0期、S期和G2期的细胞数量)。细胞转染AllStars Negative Control siRNA后,处于各细胞周期的细胞数的比例与未转染的细胞相似(参见"Normal cell-cycle distribution")。这一结果表明AllStars Negative Control siRNA没有对细胞周期产生不利影响。

报告质粒转染,确定是否整合入RISC

为了阴性对照RNAi实验的准确性,阴性对照siRNA应被整合到RISC(RNA诱导的沉默复合物)里。这意味着,阴性对照siRNA与基因特异性siRNA经过了相同的生物学过程,这使得我们能够比较使用基因特异性siRNA和阴性对照siRNA得出的两组数据,确信这是基因敲减的结果。

实验如下:

报告质粒包括一个人工合成的与AllStars Negative Control siRNA序列互补的siRNA靶向序列,该靶向序列与含有His标签的荧光报告基因相融合(参见"Reporter construct for RISC-incorporation experiment")。
该报告质粒与AllStars Negative Control siRNA和非互补siRNA共转染。同时分析未转染的细胞。
用荧光显微镜和流式细胞术检测荧光报告基因的表达。运用Western印迹通过His标签检测该融合蛋白的表达。

报告质粒和非互补siRNA共转染会导致荧光蛋白和His标签的强烈表达。当报告质粒和AllStars Negative Control siRNA共转染时,由它的互补序列将AllStars Negative Control siRNA的表达敲减,这使得整个编码荧光报告基因、His标签和siRNA目标序列的转录mRNA被降解。而mRNA的降解使得融合蛋白被敲减(参见"AllStars Negative Control siRNA is incorporated into RISC"和"Western analysis shows AllStars Negative Control siRNA enters RISC")。要实现对基因敲减的观测,必须将AllStars Negative Control siRNA整合到RISC。

Principle

转染阴性对照siRNA是每个RNA干扰试验必不可少的。阴性对照结果应该与从未转染细胞得到的结果相比较。转染阴性对照siRNA和未转染细胞的基因表达和表现型应该完全相似。如果在转染阴性对照siRNA的细胞里发现了基因表达和表现型的改变,这种改变是由转染过程或siRNA毒性和序列的非互补性引起的非特异性改变。为了确保RNAi/miRNA结果的可靠性,非特异性效应应该最小。

阴性对照结果也能与正在研究的基因特异性的RNAi/miRNA结果进行比较。这种比较使得研究者能准确描述靶基因敲减对基因表达和表现型的影响,因为阴性对照siRNA只是RNAi/miRNA的序列不同,而经历的生物学过程是完全相同的。

Procedure

AllStars Negative Control siRNA的结果可用于以下应用:

通过与未转染细胞的结果比较,可确定实验是否导致了非特异性效应 
通过与基因特异性siRNA的结果比较,可精确描述靶基因敲减的影响
在mRNA模拟实验中,通过与基因特异性miRNA模拟的结果比较,可精确描述靶基因下调的影响
Applications
  • 常规的RNAi实验
  • 起始RNAi实验
  • 高通量RNAi筛选
  • 与miRNA模拟转染有关的实验
Feature
Specifications
Design Predesigned/validated by Affymetrix GeneChip Array and cell-based assays
Format Tube
Modification Yes
Scale or yield 5 nmol, 20 nmol
Species Human, mouse, rat
Target sequence provided No

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