BLADE-R: streamlined RNA extraction for molecular diagnostics and high-throughput applications

AbstractEfficient 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. Here, we present BLADE-R, 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. 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. 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 minimal well-to-well contamination in RNA purification, cDNA synthesis, and PCR. Therefore, 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. Thus, our new BLADE-R technique works well even in low-resource environments to prepare clinical samples for PCR and sequencing experiments. It can be adapted for both low- and high-throughput formats.