HiPerFect Transfection Reagent

真核细胞的siRNA和miRNA转染
  • 低浓度条件下的siRNA高效转染
  • 高效转染原代细胞,细胞存活率高
  • 高效转染悬浮细胞和巨噬细胞
  • TransFect Protocol Database中有针对特殊细胞类型的实验方案
  • 高效转染miRNA模拟物或抑制剂
HiPerFect Transfection Reagent是一种阳离子和中性脂组成的独特混合物,能够有效摄入siRNA和有效释放细胞内部的siRNA,即使使用低浓度的siRNA也可高效敲除基因。除了siRNA,HiPerFect Transfection Reagent还适用于低通量和高通量的转染miRNA模拟物或抑制剂。TransFect Protocol Database中有适用于特殊细胞类型的实验方案。
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HiPerFect Transfection Reagent (0.5 ml)
HiPerFect Transfection Reagent for up to 166 transfections in 24-well plates or up to 666 transfections in 96-well plates
301704
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HiPerFect Transfection Reagent (1 ml)
HiPerFect Transfection Reagent for up to 333 transfections in 24-well plates or up to 1333 transfections in 96-well plates
301705
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HiPerFect Transfection Reagent (4 x 1 ml)
HiPerFect Transfection Reagent for up to 1332 transfections in 24-well plates or up to 5333 transfections in 96-well plates
301707
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HiPerFect Transfection Reagent (100 ml)
HiPerFect Transfection Reagent for transfections in up to 1388 96-well plates
301709
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The HiPerFect Transfection Reagent is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Efficient transfection of NHEK with HiPerFect Reagent.|HiPerFect Reagent provides effective CDC2 knockdown.|Reverse transfection using HiPerFect Transfection Reagent.|
Normal human embryonal keratinocytes were transfected with siRNA targeting lamin A/C or with AllStars Negative Control siRNA. Lamin A/C expression was measured by real-time RT-PCR after 48 hours.|HeLa S3 cells were transfected with a range of concentrations of siRNA targeted against CDC2 using HiPerFect Transfection Reagent from QIAGEN or Reagent L from another supplier. Non-silencing siRNA targeting green fluorescent protein (GFP) was also transfected. After 48 hours, CDC2 knockdown was assessed using quantitative, real-time RT-PCR. CDC2 expression was normalized to the expression of GAPDH. Expression levels relative to untransfected control cells are shown.|In reverse transfection protocols, cells are seeded and transfected in the same day. siRNA/miRNA is spotted into wells followed by the addition of HiPerFect Reagent. After complex formation, cells are added to the wells. These transfection protocols are rapid and convenient, can easily be automated, and are frequently used for high-throughput experiments.|
Performance

各种悬浮细胞系以及分化和未分化的巨噬细胞已进行过检测,通过使用HiPerFect Reagent进行siRNA转染检测。许多细胞系可成功转染,并有效敲减靶基因表达(见表1)。使用HiPerFect Reagent转染对于一些需电穿孔转染的细胞类型来说是最佳选择。

表1.使用HiPerFect Reagent成功转染的细胞系
细胞系 细胞类型 siRNA的浓度 敲减
K562 人慢性髓性白血病 5 nM 85%
Jurkat 人T细胞 75 nM 83%
D1.1 人T细胞 50 nM 84%
RAW 264.7 小鼠巨噬细胞 25 nM 77%
J774.A1 小鼠巨噬细胞 50 nM 97%
PMA-differentiated THP-1 人急性单核细胞白血病 5 nM 82%
MEL 鼠红白血病 5-20 nM 50-70%

HiPerFect Reagent不适合转染的细胞系(见表2)。

表2.HiPerFect Reagent不适合转染的细胞系
细胞系 细胞类型
Raji 人B细胞
U937 人巨噬细胞
Molt4 人T细胞
Molt14 人T细胞
HL60 早幼粒细胞系
E6.1 人T细胞

当siRNA的浓度在1–50 nM之间时,可有效敲除(>80%)(参见"HiPerFect Reagent provides effective CDC2 knockdown")。使用1 nM siRNA转染的敲除率为86%,而5 nM siRNA转染的敲除率可达96%。最佳的siRNA使用浓度为1 nM(最少的脱靶效应并有效敲除,参见"Efficient transfection of NHEK")或5 nM(更高的敲除效率)。

Principle

siRNA转染可能发生脱靶效应,即siRNAs影响非同源或部分同源靶基因的表达,但在很大程度上可通过使用低浓度的siRNA避免。使用HiPerFect Transfection Reagent,可高效的转染和沉默基因,在某些情况下可使用10 pM siRNA。使用HiPerFect Transfection Reagent可在很宽的siRNA浓度范围内高效转染siRNA,可使研究者选择他们所需的siRNA浓度。可用于转染多种类型的细胞,包括:HeLa、HeLa S3、HEK 293、NIH/3T3、Huh-7、HepG2、MCF-7、HUVEC和NHLF。

miRNA研究

micro RNAs(miRNAs)是一类内源性小分子RNA,与siRNAs特点相同。miRNAs在多种生物过程中有重要作用,如生长、分化和调亡。转染合成的miRNA模拟物和抑制剂,是一种用于解释特定miRNAs的靶标和作用的技术。miRNA模拟物是一种化学合成的miRNAs,模拟转染细胞的自然产生的miRNAs。miRNA抑制剂是一种单链修饰RNAs,能够特异性抑制miRNA的功能。转染miRNA模拟物抑制基因的表达,或转染miRNA抑制剂增强基因的表达,由此证明目的miRNA调节该基因的表达。而且,miRNAs在各种信号通路中的作用也可通过检查转染miRNA模拟物或抑制剂后特异性的基因表型来确定。筛查实验也可通过检查转染大分子量miRNA模拟物或混合物后的基因表型来进行。HiPerFect Transfection Reagent用于真核细胞siRNA的高效转染,也可用于miRNA模拟物或抑制剂的低通量或高通量转染。

Procedure

HiPerFect Transfection Reagent是一种即用型溶液,只需将其加入已稀释的siRNA/miRNA溶液、混合、孵育并将混合物转移到细胞即可。转染可在有血清的条件下进行,无需去除细胞中的其他物质。除了HiPerFect Transfection Reagent Handbook中的实验方案,您可以在TransFect Protocol Database中找到适合您的细胞类型和培养瓶/培养皿的实验方案。该数据库提供您所需的确切的实验方案,而不是调整已有的实验方案,节省时间和精力。只需输入细胞类型、核酸和培养瓶的类型即可打印或下载QIAGEN转染实验方案的PDF格式。TransFect Protocol Database是免费的,且无需注册。

 HiPerFect Transfection Reagent转染,可使用常规的转染步骤,在转染前24小时种细胞。同时也提供使用96孔板和384孔板反转染的实验方案(见HiPerFect Transfection Reagent Handbook)。这些实验方案,可在同一天种细胞和进行转染。加入HiPerFect Reagent后,将siRNA/miRNA加入孔中。混合均匀后,将细胞加入孔中(参见"Reverse transfection using HiPerFect Transfection Reagent")。反转染实验方案快速、方便、可实现自动化,通常用于高通量实验。

Applications

即使siRNA浓度较低,HiPerFect Transfection Reagent也能够高效进行siRNA转染,适合多种应用,如:

  • RNA干扰研究
  • miRNA研究
  • 基因表达和功能研究
Feature
Specifications
Applications RNAi studies, gene expression studies
Cell type Eukaryotic cells including primary cells
Controls Not included
Features Minimized risk of off-target effects, rapid reverse transfection
Nucleic acid siRNA, miRNA
Number of possible transfections 333 transfections in 24-well plates / 1 ml reagent
Technology Cationic and neutral lipids
Transfection type Transient transfection

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