Custom Short Hairpin RNA (shRNA) Synthesis

CD Formulation, a leading biotechnology company in nucleic acid formulation development, is dedicated to helping drug formulation researchers overcome research challenges. We offer innovative shRNA solutions and high-quality shRNA products, supported by our professional team and advanced equipment.

What is shRNA?

Short hairpin RNA (shRNA) refers to a specific sequence designed and screened to efficiently and selectively inhibit a target gene, as well as a specific structure composed of reverse repetitive sequences separated by loop sequences. This shRNA is inserted into a corresponding expression vector, such as a lentivirus, which initiates transcription in vivo to form the hairpin structure. The hairpin structure is processed in vivo by DICER and RNase, forming siRNA that induces RNA interference to inhibit specific gene expression. shRNA typically includes a transcription promoter, an shRNA precursor sequence, and a transcription termination signal.

Fig.1 Schematic of shRNA expression cassettes and processing pathways. (Sheng P, et al., 2020)

Principles of shRNA Vector Construction

• Short hairpin RNA can be cloned into expression vectors to produce small interfering RNA (siRNA) molecules. We can design shRNA sequences based on the target gene and subsequently clone them into expression vectors.
• The shRNA cloned into shRNA expression vectors consists of two short inverted repeats separated by a loop, forming a hairpin structure that is transcribed by RNA polymerase III.
• The two DNA strands are complementary and contain restriction sites at both ends for subsequent vector construction.
• The justice strand of the shRNA must have a guanine (G) at the 5' end. This is essential for ensuring that RNA polymerase can accurately recognize and transcribe it.
• The stem-loop structure of the inserted shRNA should be positioned near the center of the double-stranded RNA.
• To ensure that RNA polymerase III can terminate transcription, 5 to 6 thymidine (dT) residues must be included at the end of the shRNA insert fragment.
• There cannot be three or more consecutive T's in the justice and antisense strand sequences, as this may lead to premature termination of shRNA transcription.

Explore Our Custom shRNA Synthesis Services

Custom shRNA Services

CD Formulation's custom shRNA services enable researchers to target specific genes by designing small hairpin RNAs that effectively reduce or inhibit gene expression. These services cover sequence design, synthesis, and validation, optimizing shRNAs for cellular or in vivo experiments. They can significantly accelerate research and enhance experimental success rates in basic research, translational medicine, and drug discovery.

shRNA Vector Construction Services

Our vector construction technology ensures that at least one shRNA effectively represses the expression of the target gene. The lentiviral vectors we utilize can infect cell lines that are challenging to transfect using conventional transfection reagents, including primary cells, stem cells, suspension cells, and non-dividing cells.

General Workflow for shRNA Preparation

Fig.2 Flow chart of shRNA preparation. (CD Formulation)

• Plasmid Digestion

First, a suitable plasmid vector is selected, and the plasmid is enzymatically digested using a specific restriction endonuclease to achieve linearization.

• shRNA Synthesis

Synthesize single-stranded oligonucleotides featuring specific hairpin structures by designing short hairpin RNAs (shRNAs) based on the target gene sequences.

• Annealing

Anneal the synthesized positive and negative strand shRNA oligonucleotides to create a double-stranded structure.

• Transformation

The annealed double-stranded shRNA undergoes a ligation reaction with a linearized plasmid vector. Subsequently, the ligated product is introduced into E. coli for amplification through either chemical transformation or electrotransformation.

• Viral Packaging

Plasmids containing shRNA are introduced into packaging cell lines for the purpose of viral packaging.

• Viral Transfection

Recombinant viruses are utilized to infect target cells, thereby stabilizing shRNA expression within those cells.

• Validation

Verify the inhibitory effect of shRNA on target gene expression using qPCR or Western blot analysis.

Our shRNA Synthesis Technology Platforms

CD Formulation's shRNA synthesis platform is committed to delivering efficient and specific gene silencing tools for both research and therapeutic applications. By employing advanced synthesis technologies, we ensure the effectiveness and safety of our shRNAs.

Advantages of shRNA Synthesis Services

• shRNAs can be utilized in a wide range of cell types and biological systems, effectively targeting numerous genes for silencing. This capability positions shRNAs as a crucial tool in the research and treatment of various diseases.
• Compared to other knockout technologies, shRNAs)are relatively straightforward to design, synthesize, and manipulate. Typically, it is sufficient to clone shRNA sequences into plasmids or viral vectors and subsequently introduce them into cells to achieve the desired gene silencing effect.
• ShRNAs can induce long-term gene silencing effects in cells, with their inhibitory effects lasting for days or even weeks. This characteristic makes shRNA a valuable tool for both research and the treatment of diseases that necessitate long-term monitoring.
• Since the discovery of RNA interference technology, the design and synthesis techniques for shRNA have been continuously refined, resulting in a wide array of commercially available products. The advancement of this technology guarantees the stability and reliability of shRNAs in practical applications.

Publication Data

If you have any questions or would like more information about our shRNA services and products, please feel free to contact us.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.