Choosing the Right RNA Interference Method for your Research

RNAi also occurs in the more commonly used model system of mammalian cells, which contain conserved machinery (4). Experimental methods take advantage of the endogenous mechanism to suppress the expression of interesting genes by the exogenous introduction of nucleic acid. Some model systems accept antisense RNA or even dsRNA. However, the introduction of long dsRNA (greater than 50 base pairs) to mammalian cells induces the interferon-based anti-viral response, which causes cell death (apoptosis). Instead, experimental RNAi in the mammalian system involves the introduction of siRNA, typically 21 to 23 base pair duplexes, or small hairpin RNA (shRNA). These shRNA molecules consist of a single stand having the sequence of the two desired siRNA strands connected by non-relevant sequence. The two complementary portions anneal intra-molecularly folding the strand into a hairpin. The endogenous Dicer enzyme recognizes and cleaves the odd RNA structure into the desired siRNA molecule. Generating these two classes of molecules utilizes a variety of methods each tailored for specific applications.

2. Summary of RNAi Methods:

Chemically Synthesized siRNA

Chemically synthesized siRNA relies on the same solid-phase support chemistry used to generate DNA primers for PCR.

Expression Vectors and
Viral Vectors (including adenoviral, retroviral and lentiviral)
Containing siRNA or shRNA

Expression or viral vectors and their RNA polymerase III (Pol III) promoters drive the expression of either siRNA transcripts, as separate sense and antisense strands that anneal in the cell, or a single short hairpin RNA transcript (5-8). Human and mouse U6 and the human H1 are the most commonly used RNA polymerase III promoters. The polymerase III enzyme initiates and terminates RNA transcripts at well-defined positions (9) making its promoters well suited for the synthesis of siRNA or shRNA.

PCR-generated siRNA or shRNA Expression Cassettes and
In vitro transcribed siRNA or shRNA

The short length of these Pol III promoters (less than 300 bp) facilitates the generation of expression cassettes using PCR methods to amplify a linear fragment of double-stranded DNA containing the necessary promoters and the siRNA or shRNA sequence (10). Either the cassette itself or the purified in vitro transcript of the cassette serves as the source of nucleic acid for RNAi.

Populations of siRNA generated from RNase III or Dicer digested dsRNA

Finally, treatment of dsRNA in vitro with purified mammalian Dicer or the E. coli enzyme RNase III digests the nucleic acid into a population of siRNA duplexes. Generation of the dsRNA involves the in vitro transcription of both strands of either a gene-specific fragment or a full-length cDNA of the gene of interest cloned into an appropriate vector.

3. Delivery Methods:

Each of these classes of nucleic acid can be introduced into cells by a number of methods. In lipid-mediated transfection, cells take in non-covalent complexes between nucleic acid and a lipid or polymer reagent by endocytosis. Electroporation utilizes a brief electrical pulse to cause disruptions or holes in the cells' plasma membrane through which nucleic acid enters. Both of these methods successfully deliver any of the RNAi nucleic acids expect viral vectors. Viral vector delivery only occurs by infection of cells with the corresponding virus generated via a multi-step process. Viral vectors lack the ability to replicate themselves. Specialized cells express the missing genes necessary for viral replication and packaging. These cells produce and release virus into the culture medium upon conventional transfection with the viral vector. The virus containing the viral vector is collected and purified. Infection of the desired cell line with virus introduces the siRNA or shRNA and knocks down gene expression. The viral delivery method absolutely requires the use of viral vectors and cannot accommodate the other sources of nucleic acid for RNAi.

Successful delivery of siRNA into cells is the most crucial step in efficiently and effectively knocking down the expression of a gene of interest in cultured mammalian cells by RNAi. Several lines of evidence demonstrate that functional siRNA completely suppresses expression of the gene of interest in an individual cell as long as it enters that cell. Very sophisticated bioinformatic computer algorithms, now available, almost guarantee the functionality of the designed siRNA. Poor delivery of siRNA remains the most common reason for ineffective gene silencing. Cells vary widely in their ability to take up nucleic acid. The more readily a cell line acquires nucleic acid, the more easily siRNA silences genes in that cell. Therefore, the delivery method of choice depends greatly on the cell line of interest.

Lipid-mediated transfection, the most commonly used method, works for the most commonly used cell types, particularly any stable, secondary, transformed adherent or suspension cell line. Other commonly used cell types (such as primary cell lines, macrophages, hematopoetic cell lines and neuronal cell lines) prove very difficult to transfect with any nucleic acid using lipids. For these cell lines, electroporation provides an alternative method to deliver siRNAs into these cells. However, this method causes high rates of cell death. Besides electroporation, the viral delivery systems (lentivirus, retrovirus and adenovirus) also prove useful for the introduction of siRNA into difficultly transfected cell types, especially primary cells. Lentivirus efficiently infects dividing and non-dividing cells, but retrovirus only infects dividing cells. Adenovirus infects a broad range of cells, including dividing and non-dividing cell types.

5. Transient Versus Stable Transfection:

Nucleic acid, especially siRNA, introduced into cells generally lacks the ability to replicate itself. Therefore, the cells eventually lose the nucleic acid due to dilution by cell division. In transient transfection, assays to determine the effect of the nucleic acid (siRNA) on the cell system occur within a few days after transfection. In stable transfection, introduction of the nucleic acid of interest and an appropriate selection marker allows the selection (over the course of weeks) for the few cells that stably integrated the nucleic acid into their genome. Once expanded, these cells permanently contain the nucleic acid and express any encoding genes (or siRNA). Investigators may then perform functional assays at their leisure. Any of the RNA interference methods work in a transient transfection experiment. Typically, only vector-based systems function in stable transfections. Lentiviruses and retroviruses integrate into host genome on their own to generate stable gene silencing cell lines with high efficiency (11,12). In contrast, adenoviruses rarely integrate into the host genome; therefore, this delivery method works better to transiently silence the gene of interest (13).

Table 1 summarizes many of the generalizations that this article presents. Transient transfections of any RNAi source suffice for short-term experiments, while long-term experiments require stable transfection of expression or viral vectors. The heartier or more easily handled cells transfect with lipids or electroporation, while more fragile and difficultly handled cells require viral delivery. Therefore, the RNA interference method of choice depends on the conditions of the individual experiment.

Table 1. Summary of RNA Interference Methods

Related Products:

For transient transfections into stable, secondary, transformed adherent or suspension cells:

SureSilencing™ Human and Mouse siRNA Kits
SureSilencing™ Human and Mouse siRNA and Antibody Kits
SureSilencing™ Pathway Sets: Gene-Specific siRNA Populations

For transient transfections into stable, secondary, transformed adherent cells:

siRNA Array Plates: Six-well plates pre-coated with matrix of transfection reagent and single siRNA