GenixTalk

Operation 1: Methods of inducing RNAi and their selection

Before starting an RNAi experiment, it is necessary to choose a method suited to the purpose according to the characteristics of the gene or cell under investigation.

Cultured cells are generally used in experiments because basic data can be obtained to confirm whether the siRNA sequence selected can induce RNAi and whether the targeted gene can be easily knocked down.

There are two main ways to induce RNAi:

1] Synthetic-siRNA method
When synthetic siRNA is directly introduced into the cell, it can suppress target gene mRNA by direct action at the RNA-induced silencing complex (RISC), but it is a transient response, so that the effectiveness of the transfection reagent used is the key to success.

2] Expression-vector method
When siRNA is expressed from a vector, a steady RNAi effect can be expected once the vector is introduced into the cell. However, it takes time and effort to achieve stable expression in the cell because of the necessity of designing the shRNA, constructing the shRNA expression vector, and synthesizing the oligo-DNA.

Moreover, the most important point to note in relation to siRNA from an expression vector is that when the vector transcript is cleaved by an RNase III such as Dicer inside the cell, there is some inaccuracy at the cleavage site and the cleavage point can deviate by 1 or 2 bases, yielding an siRNA that differs from the desired product.

Whichever method is used for an RNAi experiment, it is necessary to introduce a nucleic acid into the cell. Synthetic siRNA is generally introduced into the cell by transfection. For the introduction of a vector into the cell, it is possible to induce RNAi by directly transfecting a DNA vector into cultured cells, but it is also possible to introduce it into cells by infection with a retroviral or lentiviral vector.

Operation 2: Introduction of siRNA into the cell (selection of transfection reagent and optimization of conditions)

Success in an RNAi experiment depends on the introduction of siRNA into the cell. For siRNA, a lipid-based transfection reagent is generally used, but the transfection efficiency differs greatly from one cell type to another, so it is necessary to select a suitable transfection reagent and optimize the transfection conditions for each cell type.

1] Select cell type
2] Determine culture conditions for the selected cell type
3] Select transfection reagent suited to the cells and and optimize transfection conditions.

For the optimization of transfection, the aim is to balance cell survival with siRNA uptake so as to obtain maximum uptake while maintaining a reasonable survival rate.

Operation 3: Selection of control siRNA

To accurately evaluate the knockdown effect on the target gene, it is very important to run a parallel experiment with a suitable control siRNA. And the selection of an appropriate control siRNA is key to the accurate evaluation of the knockdown of the target gene and the confirmation of the effective uptake of the selected siRNA.

1] Positive-control siRNA
A positive-control siRNA has a sequence that allows confirmation of a strong RNAi effect under suitable transfection conditions. Specifically, a positive-control siRNA allows determination of the duration and reproducibility of the RNAi effect as well as the silencing efficiency the test siRNA.

2] Negative-control siRNA
A negative-control siRNA sequence that has no complementarity to any mRNA in the target cell is selected. In particular, when a gene is targeted by siRNA in an organism, a negative-control siRNA sequence that does not exist in the genome of the organism is selected.

3] Scrambled control siRNA
Scrambled control siRNA is used as a negative-control siRNA. It has the same nucleotide composition as the test siRNA, but the sequence is randomized.

There are various ways of designing the sequence of a control siRNA, such as reversing the original sequence or inserting a mismatched nucleotide.

Operation 4: Evaluation of RNAi effect

When evaluating experimentally induced RNAi, it should be borne in mind that even among siRNA sequences designed to target the same gene, the level of RNAi activity seen depends on the sequence of the siRNA used.

Therefore, to measure the RNAi effect, it is necessary to accurately assess the expression levels of the target gene.

The RNAi effect first manifests itself as a decrease in the levels of target-gene mRNA, and then by a corresponding decrease in the expression of the encoded protein. There are various methods to assess the efficacy of gene silencing. Generally, changes in mRNA levels are measured, but changes in expression at the protein level can also be measured.

Whether mRNA levels or protein levels are measured, there are several quantitation methods. The most user-friendly, convenient, and widely used methods are as follows:

1] For measuring mRNA levels: real-time polymerase chain reaction (RT-PCR) and Northern blotting.
2] For measuring protein levels: Western blotting and enzyme-linked immunosorbent assay (ELISA).

* Genix Talk recommends that RNAi experiments be performed with high-quality siRNA synthesized with CEM™ Amidites.
We offer solutions that fit your experimental needs, including improvements in silencing efficiency, improvements in the duration of the effect within the cell, reduction in side-effects such as the interferon response, improvements in cell permeability based on the synthesis of siRNA with specific modifications or super-high-purity siRNA, and the synthesis of chimeras, as well as the synthesis of several kinds of control siRNA.

Basic knowledge of RNAi -What is RNAi?-

Discovery of RNAi

RNAi is a phenomenon in which gene expression is controlled by the sequence-specific degradation of mRNA by short interfering RNA (siRNA; 19-27-nucleotides) or double-stranded RNA (dsRNA) more than 30 nucleotides long.

Before the 1998 discovery of RNAi, antisense sequences and ribozymes were the widely used methods of controlling gene expression. However, these methods are highly dependent on the nature of the target gene and furthermore high oligonucleotide concentrations are necessary for treatment, so that the effects were insufficient to completely suppress gene expression. RNAi is gaining attention as an innovative method for overcoming these disadvantages.

RNAi is a phenomenon that exists in all organisms, including yeasts and fungi, and it is regarded as a basic phenomenon of life. Furthermore, the siRNA used in RNAi experiments is very easy to produce by chemical synthesis and shows efficient gene silencing in small amounts. The analysis of gene function made possible by the use of RNAi is expected to lead to the development of new medical treatments based on RNA itself.

Interferon response

RNAi is recognized as an effect mediated by a 19-27-nucleotide short RNA (siRNA).
In mammals, when long dsRNA (30 or more nucleotides in length) is introduced into a cell, the dsRNA-dependent protein kinase (PKR) is activated, leading to the induction of the interferon response.

The interferon response detects double-stranded RNA that has been introduced into a cell. After the induction of interferon-stimulated genes (ISG) by activated PKR, protein synthesis is suppressed and all RNA within the cell is degraded, resulting in the non-specific inhibition of gene expression and the induction of cellular apoptosis. The cell enters an antiviral state and the specific inhibition of gene expression mediated by RNAi no longer operates.

Until recently, it had been thought that the interferon response could be avoided if the RNA is kept below 30 nucleotides in length. However, it is now clear that even shorter RNA can induce the interferon response, and this remains a major problem in RNAi studies.

Mechanism of RNAi

When dsRNA is introduced into a cell, it is first cleaved into 21-23-nucleotide fragments (siRNA) by an RNase III, Dicer, after which the siRNA becomes single-stranded and is taken up by the RNA-induced silencing complex (RISC), a protein complex that directs the sequence-specific degradation of the target mRNA. Which strand of the siRNA is taken up by RISC is thought to be determined by the binding strength of the base pairs at both ends of the siRNA.

RISC recognizes the target mRNA with a sequence complementary to the siRNA single strand taken up. Then the target mRNA is cleaved by RISC and rapidly degraded. The translation of the the target mRNA into protein is inhibited and the expression of the target gene thereby suppressed.

RNAi and knockout

One widely used method of analyzing gene function involves completely eliminating gene expression by artificially deleting the target gene. This is called "gene knockout". Though the knockout method completely suppresses target-gene expression, it has some disadvantages such as the time and effort required to obtain gene-analysis results. So gene knockout cannot be seen as a complete solution for gene analysis.

In contrast to this, provided siRNA or dsRNA can be efficiently introduced into the cell, RNAi can quickly inhibit target gene expression. Because the induction of RNAi makes it possible to control gene expression without damaging the gene itself, it is called "gene knockdown" as opposed to "gene knockout". These two methods have their own merits and demerits. Gene knockout is an effective conventional method of gene analysis, but these days gene knockdown is widely used because it simplifies the process of gene analysis.

Advantages and disadvantages of gene knockdown and gene knockout

Advantages of gene knockdown:

• The induction of RNAi is simple.
• It is possible to analyze genes whose deletion by knockout is lethal.
• There is no damage to the gene itself.
• Specific inhibition of gene expression is possible.

Disadvantages of gene knockdown:

• It cannot completely suppress gene expression.
• There is the possibility of the induction of an interferon response.
• There is the possibility of off-target effects.
• The efficiency of suppression of gene expression depends on the sequence of the siRNA.

Advantage of gene knockout:

• Complete suppression of gene expression is obtained

Disadvantages of gene knockout:

• Time and effort are needed to produce the knockout organism.
• It is hard to analyze genes whose deletion is lethal.
• It is difficult to apply to organisms for which the knockout technology has not yet been established.