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Introduction

Short-hairpin RNA (shRNA) molecules are the precursors and effective triggers of RNA interference (RNAi) in most organisms. Until now shRNA molecules are usually produced through fermentation by engineering bacteria. For instance, E. coli strain HT115 (DE3) was chosen to take advantage of an IPTG-inducible T7 RNA polymerase gene to produce shRNA molecules. However, this production method has the disadvantages of low yields and high cost. Therefore, it is rather difficult for RNAi to be widely applied in the field of agricultural due to the high production cost of shRNA molecules. Recently, advances in RNA biology demonstrated efficiently the cell-free method of large-scale synthesis of shRNA molecules. It is impressing that low cost and high-efficient shRNA production method could be established through in vitro transcription. Consequently, we have undertaken the design and construction of an automated hardware system tailored for the large-scale synthesis of shRNA molecules. This endeavor aims to enhance the convenience of shRNA production while concurrently reducing associated costs. Our innovative and cost-effective equipment exhibits several advantages when compared to the traditional, more expensive PCR instruments.

Principles of cell-free production of shRNA——in vitro transcription

In vitro transcription is a technique used to synthesize RNA molecules from a DNA template outside of a living cell (Fig 1). In brief, T7 RNA polymerase synthesizes RNA complementary to the DNA strand from downstream of the template DNA T7 promoter (the green box), obtaining a large number of RNA molecules rapidly and cost-effectively.

Fig 1. The principles of in vitro transcription.

The protocol of cell-free production of shRNA molecules:

• (1) Mix 1.2mL T7 Enzyme Mix (contains T7 RNA polymerase, NTPs and Transcription Buffer) with 0.8mL DNA template (prepared by PCR amplification or chemical synthesis)
• (2) Incubate at 37 ℃ for 2h.
• (3) Add 2mL dual enzyme digestion system (contains DNase I and RNase T1) to the reaction system.
• (4) Incubate at 37 ℃ for 30min.

Components and constructions of our hardware

Generally, the hardware is composed of 9 different elements. Their specific types and functions are presented in detail in the following table (Fig 2). Specifically, 3 peristaltic pumps coupled with their corresponding drives facilitate the precise transport of a specified volume of reagents. The speed of the peristaltic pumps could be accurately controlled through enabling the microcontroller pins to emit pulse width modulation (PWN) waves with specific duty cycles, thus regulating the rates of flow of the reagents within the hose. It could be inferred from the table that the total cost of the equipment amounts to approximately $191, significantly lower than that of a traditional PCR instrument. Thus, it is promising to reduce the production costs associated with shRNA molecules by utilizing our equipment.

Fig 2. The summarization of the components used in the hardware.

Furthermore, to establish large-scale production of shRNA molecules, we designed and 3D-printed a high-capacity disposable reaction container (Fig3). This innovative reaction container was designed by 3DsMAX software and 3D-printed with heat-resistant resin material. Prior to use, the reaction container underwent thorough sterilization. Our designed reaction container is capable of a total volume of 4mL reaction reagents, which is 20 times larger than that of a single centrifuge tube (0.2mL) commonly employed in traditional PCR instruments. Hence, our built hardware demonstrates the capability to scale up the production of shRNA molecules substantially.

Fig 3. The 3D modeling drawing of the reaction container(A) and the physical picture of the 3D-printed reaction container(B).

Additionally, the construction of the equipment is detailed as follows (Fig 4, Fig 5). The essential reagents for the in vitro transcription reaction were prepared in three separate centrifuge tubes. Each pump was connected to an interface on the reaction container and its corresponding centrifuge tube through silicone hose, facilitating the precise transfer of a specified volume of reagents from the centrifuge tube into the reaction container. The positive temperature coefficient (PTC) thermostatic heater was sticked to the bottom of the reaction container, while the temperature probe was inserted into the reaction container through the hole on its top. Within this reaction container, the in vitro transcription reaction takes place.

Fig 4. The sketch map of the hardware.

Fig 5. The physical picture of the hardware.

Schematic diagram

The schematic diagram of the hardware was designed and drew by Altium Designer, which consists of the circuit schematic design of microcontroller, temperature sensor, drive of peristaltic pump and drive of PTC heater. Both the drive of peristaltic pump and the drive of PTC heater are made up of superpower MOS transistor control circuit.

Fig 6. The schematic diagram of the hardware.

Working procedure of the equipment

Fig 7. Working procedure of the equipment.

This working procedure is achieved by the C program uploaded into the microcontroller according to the protocol of cell-free production of shRNA molecules. The source code of the program is provided below. It is noticeable that the equipment establishes automatic production of shRNA molecules through microcontroller programming, enabling it to manufacture shRNA molecules conveniently in the absence of human manual effort. Consequently, the equipment can be operated by non-scientific research personnel or utilized in a non-laboratory setting.

Production result of the equipment

The shRNA molecules produced by the equipment were examined using nucleic acid electrophoresis. To compare the shRNA synthesis, DNA templates (approximately 100 bp in size) used for shRNA synthesis were loaded onto the same gel. It could be inferred from the results that the content of synthesized shRNA is higher than that of the DNA templates. In conclusion, the automated hardware system offers a cost-effective, user-friendly, and scalable solution for shRNA molecule synthesis, making RNA interference technology more accessible and efficient across various applications.

Fig 8. Agarose gel depicting the synthesized shRNA and comparing it with the DNA templates used for shRNA reaction.

Comparison of our equipment with traditional PCR instrument

Fig 9. Comparison of our equipment with traditional PCR instrument.

User product manual

In order to let users understand the operation process of the hardware, we have written very detailed user product manuals.

Presentation video of the hardware: