I. Differences Between siRNA and shRNA

1. Structure and Composition

siRNA (Small Interfering RNA): siRNA is a double-stranded RNA molecule, typically 20-25 base pairs in length, with a 2-nucleotide 3' overhang at both ends. It consists of two complementary RNA strands: a sense strand (homologous to the target mRNA) and an antisense strand (complementary to the target mRNA). siRNA is usually generated from exogenous or endogenous double-stranded RNA through cleavage by the Dicer enzyme.

shRNA (Short Hairpin RNA): shRNA is a single-stranded RNA capable of forming a hairpin (stem-loop) structure, comprising a short double-stranded region (19-29 bp) and a loop region. It consists of a sense strand, a loop sequence, and an antisense strand, forming a hairpin shape. shRNA is constructed into expression vectors through genetic engineering techniques and forms a hairpin structure upon transcription.

2. Mechanism of Action

siRNA: After being cleaved by the Dicer enzyme, siRNA directly binds to the RNA-induced silencing complex (RISC). The antisense strand of siRNA guides RISC to recognize and cleave the target mRNA complementary to the siRNA, leading to mRNA degradation.

shRNA: After being transcribed from an expression vector, shRNA is cleaved by the Dicer enzyme into siRNA, which then enters RISC to exert its function. shRNA first forms a hairpin structure, which is subsequently processed into siRNA, ultimately resulting in the degradation of the target mRNA.

3. Application Methods

siRNA: siRNA is typically obtained through chemical synthesis or in vitro transcription and is directly transfected into cells. It is suitable for short-term experiments, as it can exert its effects within hours of transfection, but the duration of its interference effect is relatively short (usually a few days).

shRNA: shRNA is introduced into cells via viral vectors (e.g., lentivirus, adenovirus) or plasmid vectors, integrating into the genome to achieve long-term expression. shRNA is continuously transcribed within cells, generating siRNA, and is suitable for long-term experiments or the establishment of stable cell lines.

4. Advantages and Disadvantages

siRNA

Advantages: Simple operation, suitable for rapid screening. Avoids potential risks associated with genomic integration.

Disadvantages: The interference effect is short-lived, requiring repeated transfections. Chemical synthesis is costly.

shRNA

Advantages: Enables long-term, stable gene silencing. Suitable for in vivo experiments and animal models.

Disadvantages: The process of vector construction and viral packaging is complex. There is a risk of genomic integration, potentially leading to insertional mutagenesis.

II. Group Settings for Conventional Interference Detection

Reasonable group settings are crucial for ensuring the reliability of experimental results in RNA interference studies. Below are recommendations for conventional interference detection groupings:

1. Experimental Group

Transfect cells with siRNA or shRNA specific to the target gene.

Different concentrations or time points can be set to observe the interference effect.

2. Control Groups

Negative Control Group (NC): Transfect cells with non-specific siRNA or shRNA that has no homology to the target gene. Used to exclude non-specific interference effects.

Mock Control Group: Do not transfect any RNA or vector; only add transfection reagent. Used to assess the impact of the transfection reagent on cells.

3. Positive Control Group (Optional)

Known Effective Interference Group: Transfect cells with siRNA or shRNA targeting a known easily interfered gene. Used to validate the experimental system and transfection efficiency.

4. Summary of Group Settings

The experimental group is used to verify the interference effect on the target gene.

The negative control group is used to exclude non-specific interference.

The mock control group is used to assess the impact of the transfection reagent.

The positive control group (optional) is used to validate the effectiveness of the experimental system.

5. Precautions

Repetition of Experiments: Set at least three biological replicates for each group to reduce experimental error.

Detection Indicators:

mRNA Level: Detect the mRNA expression level of the target gene using qRT-PCR.

Protein Level: Detect the protein expression level of the target gene using Western blot or ELISA.

Phenotypic Changes: Observe changes in cell proliferation, apoptosis, migration, etc.

Time Point Settings: Set multiple time points (e.g., 24 hours, 48 hours, 72 hours) based on the gene expression cycle and experimental objectives.

III. Summary

Differences Between siRNA and shRNA:

siRNA is a double-stranded RNA that directly exerts its function; shRNA is a single-stranded RNA that forms a hairpin structure and is then processed into siRNA.

siRNA is suitable for transient interference, while shRNA is suitable for stable interference.

Group Settings:

It is essential to set up an experimental group, a negative control group, and a mock control group. A positive control group can be added if necessary.

Ensure the reliability and reproducibility of results through multiple time points and repeated experiments.

By employing reasonable group settings and scientific experimental designs, the effectiveness of RNA interference can be effectively verified, providing a reliable basis for subsequent research.