Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)

Abstract

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.

This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.

Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.

In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.