Antisense Oligonucleotide (ASO) In Vitro Screening Service

Introduction

Antisense Oligonucleotides (ASOs) are a significant research and therapeutic tool, providing strong support for exploring gene function, and disease mechanisms and developing new therapeutics.

After the design, synthesis, and production of the ASO sequence, it is necessary to adopt a set of well-designed in vitro experiments, for activity verification and functional verification of the designed ASOs, including mRNA level inhibition efficiency detection and protein expression level evaluation. Creative Biolabs can provide professional ASO in vitro screening services to provide customers with efficient, accurate, and customized experimental solutions.

Screening Strategies Based on in vitro Cell Models

ASO efficiency can be analyzed in various cell models in vitro:

Primary cells and iPSC-derived disease models: Primary cells are cells obtained directly from organisms and have a high degree of physiological relevance and representation. iPSC-derived disease models mimic disease states by reprogramming a patient's somatic cells into induced pluripotent stem cells, which then differentiate into specific types of cells or tissues. Creative Biolabs will provide ASO efficiency detection services in primary cells and iPSC-derived disease models, including cell culture, transfection, detection, and other steps.

Other cell lines:

Organoids: Organoids grown in a three-dimensional environment more closely resemble the organization and physiology of natural epithelial cells than 2D cultured cells. ASO can be taken up efficiently by organoids without transfection reagents, making it a convenient tool for routine laboratory research and screening¹.

Notes:

• Cells with high expression of target genes were selected to verify the silencing efficiency of ASO.
• Normal and pathological cells (such as tumor cells) were selected to evaluate the difference in ASO activity under normal and pathological conditions.
• Target gene knock-out or knock-down models were used as positive controls.

In the process of cell culture, the following aspects need to be paid attention to:

Detection of Gene Silencing Efficiency

• RT-PCR

RNA is extracted from ASO-treated cells or tissues, and the expression level of target RNA is determined using techniques such as real-time reverse transcription-PCR (qRT-PCR) to evaluate the gene-silencing effect of ASO.

Fig.1 Schematic diagram of RT-PCR procedure and analysis of amplification curve.²

• Western Blotting

Western Blotting can be used to detect the expression level of target proteins. By comparing the protein expression levels before and after ASO treatment, the knockout efficiency of ASO can be evaluated.

• Flow Cytometry

Flow cytometry can detect the expression level of specific proteins in cells or on the cell surface, evaluate the knockout effect of ASO, and analyze the correlation between various indicators.

Fig.2 Working principle of flow cytometer.Distributed under Open Access license CC BY 4.0, from Wiki without modification.

• Cell Function Experiment

According to the function of the silenced target genes, cell function tests such as MTT, CCK8, CTG, cell invasion, and scratch tests can be performed for cell cycle, cell proliferation, apoptosis, and cell killing efficacy.

• Reporter gene assay

A reporter gene system containing a target RNA sequence was constructed to evaluate the knockout efficiency of ASO. Creative Biolabs offers mature luciferase reporting systems and dual luciferase reporting systems construction, detection, and analysis services.

Analysis of detection results

Efficient silencing: If the experimental results show that the expression levels of target mRNA and target protein are significantly reduced, and an ASO is evenly distributed in the cell, then the ASO can be considered to have an efficient silencing effect.

Specificity: By comparing the silencing effect of different sequences of oligonucleotides on target genes, the specificity can be assessed.

Mechanism of action: Based on the distribution of oligonucleotides in the cell, the mechanism of action can be reasonably speculated.

Highlight

Creative Biolabs uses advanced bioinformatics tools to design efficient, highly specific antisense oligonucleotide sequences based on information provided by the customer. At the same time, multiple design strategies can be provided to verify the function of ASO, including mRNA level inhibition efficiency detection and protein expression level assessment. Provide detailed laboratory reports, including experimental methods, data results, and statistical analysis. If you have any questions or needs regarding ASO in vitro screening services, please feel free to contact us and we will be glad to serve you.

References

1. Lange, Jenny, Haiyan Zhou, and Amy McTague. "Cerebral organoids and antisense oligonucleotide therapeutics: challenges and opportunities." Frontiers in Molecular Neuroscience 15 (2022): 941528.
2. Wong, Marisa L., and Juan F. Medrano. "Real-time PCR for mRNA quantitation." Biotechniques 39.1 (2005): 75-85. Distributed under Open Access license CC BY 4.0, without modification.