NanoVIP® Automates Manual RNAscopeTM ISH Protocol

Background   

OmicsVeu is pleased to announce one more achieved milestone i.e., automating manual RNAscope^TM process through an all-in-one NanoVIP^® autostainer. The overall manual protocol can easily be automated precisely and efficiently by reducing hands-on time and cost saving manner.

RNAscope^TM is proprietary technology that uses RNA in situ hybridization (ISH) technique to locate RNA transcripts in a sample. Traditional transcriptomic studies involved study gene expression by first homogenizing tissues or cells as a single unit. Thus, information about single cells or heterogeneity of cells is lost. The RNAscope^TM method enables scientists to further study and understand transcriptomic heterogeneity of cells in a population,and the microenvironment, and even mechanisms of therapy-resistance or residual disease in cancer.

RNAscope^TM Workflow

RNA probes are first designed to target specific RNA sequence of interest (Figure 1). Of note is the dual-Z RNA probes special to RNAscope^TM. Z probes are designed to hybridize in adjacent in pairs. This is the only configuration where a pre-amplifier can then bind to the R probes. Pre-amplifiers cannot bind to single Z RNA probes. Amplifiers then bind to the pre-amplifier. Amplifiers, as the name suggests, provide multiple binding sites for hybridization of signaling molecules such as chromatin, fluorophores, or metal-oligos.

The overall workflow of RNAscope^TM consists of 5 steps (Figure 2).

• Permeabilization—Samples (cells or tissue) are fixed onto slides and various treatments performed to unmask target RNA and prepare cells and tissues for further hybridization steps.
• Hybridization—Targeted dual-Z probes are added to the slides and hybridized to target RNA sequences.
• Amplification—Amplifiers provide points of hybridization of label probes
• Visualization under microscope or other imagining system
• Quantification analysis using manual counting or other imaging analysis software

RNAscope^TM technology is yet another technique in our arsenal to study cellular transcriptome. However, the first three steps in the workflow (permeabilization, hybridization, and amplification) can take 3 hours of hands-on time from an individual who is knowledgeable and highly skilled. For new technicians or individuals who are not knowledgeable with laboratory practices, this protocol may be more challenging. At Omicsveu, we aim to simplify and streamline manual RNAscope^TM staining procedure by automation with NanoVIP^®.

Automation with NanoVIP^®

NanoVIP^®’s fully automated staining workflow streamlines the manual RNAscope^TM protocol leading to reduction of costs by decreasing hands on time and reagent volumes used. One of the key advantages of NanoVIP^® baking, dewax, and antigen retrieval steps are all performed using NanoVIP^® along with other staining procedures. There is no need for an additional instrument. Another advantage includes the precision pipetting arm along with proprietary micro-chamber slide technology which minimizes reaction volumes. Below are more key features and benefits of NanoVIP^® along with the automated workflow (Figure 3).

Key Features

• All steps are performed by NanoVIP^®—Hands-on steps involve mixing the appropriate reagents and solutions for loading;
• Estimated hands-on time: < 1 hour—This time is an estimation of collecting and filling reagent vials and buffers;
• Total assay time reduced from > 10 hours to < 7 hours;
• Once users load reagents, buffers, and slides into NanoVIP^®, there is no more hands-on time until the final removal of slide for imaging;
• 150 μl of probe volume needed for NanoVIP^® instead of 300 μl of manual requirements.

Advantages

• Cost-effectiveness: Reducing reagent costs and labor compared to conventional methods;
• Time-efficiency: Automating manual staining steps saves considerable time and generates consistent results with antigen retrieval done within the instrument; and
• Streamlined Process: Using automated process simplifies the protocol, making it more accessible to researchers.

Conclusion

The RNAscope^TM staining system amplifies targeted RNA transcripts and allows for visualization on the tissue and cellular level. However, manual protocols require more hands-on time, higher reagent volumes, and more variation in results due to manual pipetting and handling. Omicsveu has automated RNAscope^TM manual protocol using NanoVIP^® and reduced hands-on time from > 3 hours to < 1-hour, overall assay time from > 10 hours to < 7 hours, and antibody volumes from 300 μl to 150 μl while ensuring consistency with clean, crisp, and intense staining results.

A comparison between RNAscope^TM manual protocol and the automation of NanoVIP^® is illustrated below.

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