Introduction

Small Interfering RNAs (siRNAs) are short double-stranded RNA molecules that can target and degrade complementary mRNAs via a cellular process termed RNA interference (RNAi). Alternate methods for generating siRNA are: (i) vector based in vivo expression, (ii) chemical synthesis, (iii) in vitro transcription (IVT), (iv) RNAse III mediated hydrolysis and (v) PCR based siRNA expression cassettes. We have evaluated these diverse methodologies and found them to offer varying advantages over one another.

简介

小干扰RNA(siRNA)是一种短双链的RNA分子,可以在细胞内靶向结合并降解互补mRNA,这一过程被称为RNA干扰(RNAi)。产生siRNA的方法有:(i)载体体内表达,(ii)化学合成,(iii)体外转录(IVT),(iv) RNAse III介导的水解过程,以及(v)基于PCR的siRNA表达组件。我们对这些不同方法进行了评估,发现它们相互之间各具优势。

Figure 1. Use of Chemically Synthesized and in Vitro Transcribed siRNAs to Induce Gene Silencing. siRNAs targeting ß-Actin were prepared by chemical synthesis (Ambion) or by in vitro transcription using Ambion's Silencer™ siRNA Construction Kit. HeLa cells were plated at 30,000 cells per well in a 24 well tissue culture plate containing glass slides. The cells were transfected 24 hours after plating, using 2 µl siPORT™ Lipid (Ambion) according to the manufacturer's protocol, at a final siRNA concentration of 75 nM. Immunofluorescence analysis was performed 96 hr post transfection using mouse anti-Human ß-Actin primary antibody and a FITC conjugated anti-mouse IgG secondary antibody. Photographs were taken using the appropriate fluorescent filters and quantified using MetaMorph software. Note that both siRNA preparation methods resulted in >_ 95% reduction in ß-actin protein levels.

 

Figure 2. Schematic of a Typical siRNA Expression Vector. A. Vector Diagram. B. siRNA encoding insert. C. Hairpin siRNA.

 

Figure 3. Long Term Silencing of GFP with pSilencer™ 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP or pSilencer 2.1-U6 hygro without an siRNA-encoding insert. Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.

 

Figure 4. siRNA Expression Plasmids Under Transient Selection to Induce Gene Silencing. HeLa cells were plated at 60,000 cells per well and then co-transfected, using 1.2 µl FuGENE 6 (Roche) according to the manufacturer's protocol, with 400 ng GFP-expressing plasmid and 400 ng of each of 6 different siRNA expressing plasmids containing a selectable marker (Ambion) and encoding an siRNA targeting GFP. The cells were placed under selection at 24 hours post transfection using 300 µg/ml Hygromycin, 2.5 µg/ml Puromycin, or 2000 µg/ml G418. At 48 hours post transfection, the antibiotic containing media was removed and replaced with normal growth media. At 72 hours post transfection, the cells were harvested and analyzed. Blue: DAPI stained nuclei. Green: GFP.

 

Figure 5. Silencing of Gene Expression by PCR-based siRNA Expression Cassettes. A. Schematic representation of the PCR strategy used to yield the PCR-based siRNA expression cassettes [Castanotto et al., (2002) RNA 8:1454]. B. Inhibition of exogeneous EGFP expression using siRNA-encoding PCR cassettes. HeLa cells were co-transfected with PCR cassettes encoding an EGFP or scrambled control siRNA and a target EGFP expressing plasmid and analyzed by fluorescence microscopy. High levels of EGFP expression can be detected in control cells, but not in cells transfected with EGFP siRNA-encoding PCR products. C. Inhibition of endogeneous human GAPDH expression using siRNA-encoding PCR cassettes. PCR cassettes encoding a human GAPDH and a scrambled control siRNA were transfected into HeLa, DU145, and HEK 293 cells and analyzed by Northern Blot. GAPDH expression levels were reduced by >80% in cells treated with GAPDH siRNA-encoding PCR cassettes over control cells.

 

Figure 6.  Comparison of siRNAs Prepared by Chemical Synthesis vs RNase III Digestion of Long dsRNA. A population of siRNAs targeting GAPDH was prepared by synthesizing and purifiying a 200 bp dsRNA, corresponding to the first 200 bases downstream of the AUG start site of GAPDH mRNA, with the MEGAscript RNAi Kit (Ambion) and then cleaving 15 µg of the transcript with 15 U RNase III (Ambion; Panel A). HeLa cells were plated at 30,000 cells per well in a 24 well tissue culture plate on glass cover slips. The cells were transfected 24 hours later with 100 nM, 50 nM, 25 nM, or 12.5 nM final concentration of the siRNA population or a chemically synthesized siRNA known to efficiently silence GAPDH. The cells were harvested after 48 hours and the reduction in protein levels was examined by immunofluorescence microscopy. The level of GAPDH in the cells was reduced efficiently by siRNAs prepared by both chemical synthesis and RNase III digestion.

 
  Chemical synthesis RNase III digestion of dsRNA siRNA Expression Vector PCR Expression Cassette In vitro transcription
Requirements 2 21-mer RNA oligos Transcription template (200-800 bp region flanked by T7 promoters) 2 55-60-mer DNA oligos 2 ~55-mer DNA oligos 2 29-mer DNA oligos
Turnaround time (total preparation/synthesis time) 4 days to 2 weeks* 1 day + transcription template preparation time 5+ days + DNA oligo ~ 6 hours + DNA oligo 24 hours + DNA oligo
Hands on time Little to none* Moderate High Moderate Moderate
Testing to find optimal siRNA sequence Required Not needed Required Required Required
Ability to label siRNA (i.e., for analyzing siRNA uptake or localization by fluorescence microscopy)     No No Yes
Relative ease of transfection Good   Fair Good Good
Selectability (i.e, antibiotic selection)   No      
Useful for long term studies No No Yes, with selection No No
Ability to scale up synthesis Yes Limited Yes Limited Limited
Monitor transfection efficiency of entire population No No Yes No No
Relative cost per gene (not including labor) High Low Moderate Moderate Moderate

*Depends on purification/deprotection options selected and format (e.g., annealed and ready to transfect versus single strands supplied lyophilized)

图1. 使用化学合成或者体外转录的siRNA来诱导基因沉默。通过化学合成方法(Ambion)或体外转录方法(使用Ambion Silencer™ siRNA Construction Kit)制备靶向ß-Actin的siRNA。在带有载玻片的24孔组织培养板上对HeLa细胞进行平板培养至每孔30,000个细胞。细胞铺板24小时后,按照制造商实验流程使用2 µl siPORT™ Lipid (Ambion) 进行转染,siRNA最终浓度为75 nM。转染后96小时使用小鼠抗人ß-Actin一抗及FITC标记的抗小鼠IgG二抗进行免疫荧光分析。使用相应的荧光滤镜拍摄得到图像并使用MetaMorph软件进行量化。结果表明两种siRNA制备方法均导致ß-actin蛋白水平降低了>_ 95%。

 

图2. 典型siRNA表达载体图解。A、载体图谱。B、siRNA编码插入片段。C、发卡结构siRNA。

 

图3. 使用pSilencer™ 2.1-U6 hygro对GFP进行长效沉默。分别使用含编码siRNA(靶向cycle 3 GFP)插入片段的pSilencer 2.1-U6 hygro载体或不含编码siRNA插入片段的载体转染至表达cycle 3 GFP的HeLa细胞。在转染之后,对细胞进行潮霉素筛选。筛选三周后,使用荧光显微镜对细胞中GFP的表达进行分析。绿色:GFP。蓝色:DAPI染的细胞核。与转染了“空”siRNA表达载体的细胞相比,转染了编码GFP siRNA的pSilencer 2.1-U6 hygro siRNA表达载体的细胞的GFP水平发生了显著下降(94%)。

 

图4. 利用siRNA表达质粒通过瞬时筛选诱发基因沉默。HeLa细胞分板至每孔60,000细胞,然后进行共转染:根据制造商实验流程,使用1.2µl FuGENE 6 (Roche),以及400ng的GFP表达质粒和400ng的6种不同siRNA表达质粒中的一种进行转染,其中每种siRNA表达质粒均含有一种筛选标记物(Ambion),并且均编码靶向GFP的siRNA。在转染24小时后使用300 µg/ml潮霉素、2.5 µg/ml嘌呤霉素或2000 µg/ml G418对细胞进行筛选。转染48小时后,使用正常生长培养基替换含抗生素的培养基。转染72小时后,收集细胞并进行分析。蓝色:DAPI染色细胞核。绿色:GFP。

 

图5. 使用基于PCR的siRNA表达组件进行基因表达沉默。A、用于得到基于PCR的siRNA表达组件的方法图解[Castanotto et al., (2002) RNA 8:1454]。B、使用编码siRNA的PCR组件抑制外源EGFP表达。将编码EGFP的质粒与编码随机siRNA的PCR组件或者针对EGFP的siRNA PCR组件共转染到HeLa细胞中,然后使用荧光显微镜进行分析。在对照细胞中可检测到高表达水平的EGFP,而在转染了编码EGFP siRNA的PCR产物的细胞中则没有出现这一现象。C、使用编码siRNA的PCR组件抑制内源性人GAPDH表达。将编码人GAPDH或者随机对照siRNA的PCR组件转染至HeLa、DU145及HEK293细胞,然后使用Northern Blot进行分析。相比对照细胞,经编码GAPDH siRNA的PCR组件处理的细胞中,GAPDH表达水平降低>80%。

 

图6. 化学合成方法制备的siRNA与RNase III消化长链dsRNA得到的siRNA的比较。使用MEGAscript RNAi Kit (Ambion),合成并纯化200 bp长的dsRNA来制备靶向GAPDH的siRNA群,其序列对应于GAPDH mRNA的AUG起始位点下游的前200个碱基,然后使用15 U的RNase III (Ambion; Panel A) 对15 µg转录本进行剪切。使用带玻璃盖片的24孔组织板对HeLa细胞分板,每孔30,000个细胞。24小时后对细胞使用100 nM、50 nM、25 nM或12.5 nM终浓度的siRNA群或化学合成的已知可有效沉默GAPDH的siRNA进行转染。48小时后收集细胞并使用免疫荧光显微镜对蛋白水平变化进行检测。通过化学合成及RNase III消化制得的siRNA均可有效降低细胞中的GAPDH水平。

 
  化学合成 dsRNA的RNase III消化 siRNA表达载体 PCR表达组件 体外转录
必需原料 2条长度21个nt的RNA寡核苷酸 转录模板(长度约200-800 bp,两侧分别有一个T7启动子) 2条长度为 55-60 nt的DNA寡核苷酸 2条长度约为 55nt的DNA寡核苷酸 2条长度约为 29nt的DNA寡核苷酸
总共时间(制备/合成总时间) 4天至2周* 1天+转录模板制备时间 5天+DNA oligo ~ 6小时 + DNA oligo 24小时 + DNA oligo
操作时间 少或无* 中等 中等 中等
最优siRNA序列的鉴定测试 需要 不需要 需要 需要 需要
可否对siRNA进行标记(例如:可以使用荧光显微镜分析siRNA摄取或定位)     不可以 不可以 可以
转染相对难易程度 简单   正常 简单 简单
可否筛选(即抗生素筛选)   不可以      
可否用于长期研究 不可以 不可以 可以,需要进行筛选 不可以 不可以
能否大规模合成 可以 受限 可以 受限 受限
监控转染效率 不可以 不可以 可以 不可以 不可以
每条基因相对成本(不含人力成本) 中等 中等 中等


*取决于所选择的纯化/去保护方法及规格(例如退火后即可进行转染/单链冻干形式)

小结

Vector based in vivo expression:

  • Permits long-term and stable gene silencing
  • Possibility to use inducible/repressible markers
  • Use of viral vectors

Chemical and IVT synthesis:

  • Allows rapid screening of multiple targets
  • High siRNA homogeneity and purity

RNase III mediated hydrolysis:

  • Eliminates the need for screening of target site
  • Overcomes variability in silencing by synthetic/IVT siRNA
  • Cost-effective for functional genomic studies

PCR based siRNA expression cassettes:

  • Ideal for screening siRNA sequences prior to cloning in a vector
  • Rapid and inexpensive procedure

基于载体的体内表达:

  • 可进行长期稳定的基因沉默
  • 可使用诱导性/抑制性标记物
  • 可使用病毒载体


化学及IVT合成:

  • 可对多个目标进行快速筛选
  • 较高的siRNA均一性及纯度


RNase III介导的水解:

  • 无需筛选靶向位点
  • 克服了合成/IVT siRNA沉默时的可变性
  • 对于功能基因组研究而言性价比较高


基于PCR的siRNA表达组件:

  • 适于在克隆至载体前对siRNA序列进行筛选
  • 实验步骤快速、便宜