B-247 BLADE-R: streamlined RNA extraction for clinical diagnostics and high-throughput applications

Abstract Background Efficient nucleic acid extraction and purification are crucial for cellular and molecular biology research, yet they pose challenges for large-scale clinical RNA sequencing and PCR assays. As most molecular biology assays begin with the separation of DNA, RNA, and/or protein there has been a worldwide, concerted effort to increase the quality and yield of biomolecular extractions for almost two centuries. However, as Polymerase Chain Reaction (PCR) and DNA/RNA sequencing-based assays become more clinically relevant, there is increasing pressure for more streamlined, and high-throughput, lab methods for nucleic acid extraction and processing to meet clinical demands. Selecting the right nucleic acid extraction technique is crucial since it affects the precision of the molecular diagnosis. We have developed a novel BLADE-R, (Bead Lysis and DNA Elimination for RNA Extraction), a magnetic bead-based protocol that simplifies the process by combining cellular lysis and nucleic acid binding into a single step, followed by a unique on-bead rinse for nuclease-free separation of genomic DNA and RNA. Methods We employed our novel Blade-R approach for RNA extraction and compared it to traditional TRIzol method of RNA extraction. Our method comprises a one-step sample lysis and binding of nucleic acid to magnetic beads. Two wash steps and two RNA precipitation steps result in pure RNA yield. We have tested our method on several samples such as cell lines, frozen human tissues and clinical blood samples. We assessed our sample quality by nanodrop and Agilent TapeStation. RT-qPCR and RNA sequencing were performed to assess the utility of our extracted RNA for downstream applications. We developed a high-throughput method to extract RNA from clinical blood samples for RT-qPCR and RNA sequencing suitable for clinical applications and diagnostics Results The Agilent TapeStation and RT-qPCR analyses show that RNA extracted from HEK293T cell line using BLADE-R outperforms the TRIzol protocol in terms of time and cost. RNA sequencing reveals no differences in sequence quality or gene count variance between samples processed with BLADE-R and those processed with TRIzol followed by RNA kit clean-up. These results suggest that RNA purified with BLADE-R can be used to produce high-quality RNA sequencing library preps without introducing any detectable sequencing bias. Additionally, BLADE-R outperformed TRIzol in RNA extraction from frozen tissue and whole blood samples, as confirmed by RT-qPCR. Our protocol can be adapted to a 96-well plate format, enabling RNA purification of up to 96 human blood samples in less time than a single-sample traditional extraction. Using BLADE-R in this format, we confirmed no well-to-well contamination in RNA purification, cDNA synthesis, and PCR. Conclusion Our novel BLADE-R protocol, suitable for both low and high-throughput formats, is effective even in limited-resource settings for preparing clinical samples for PCR and sequencing assays. This method enables rapid, high-yield RNA extraction while minimizing contamination and reducing processing time. The BLADE-R technique is suitable for automation, in addition to manual high-throughput RNA isolation. Therefore, our novel BLADE-R technique is particularly suited for clinical and high/low-resource settings, offering a scalable and cost-effective solution for high-throughput RNA purification and do