Abstract B44: An optimized method of multimodal mRNA and gDNA isolation from low biomass input

Background: In cell-based applications in liquid biopsy research (circulating tumor cells [CTC], fetal cells, endothelial cells), often transcriptomes get analyzed. Here and in single-cell research there is a requirement to also investigate genomic variants. Therefore, a multimodal sample preparation for both gDNA and RNA (preferably mRNA) is increasingly needed in the market. There are several combined RNA/DNA preparation solutions already available with the following caveats: Optimizing DNA yield and quality is often achieved under the compromise of accepting losses in RNA yield and/or quality or vice versa. This may be acceptable for larger sample inputs but is critical for low-biomass samples. The goal of this project, therefore, was to develop an optimized AllPrep solution for low-biomass samples that provides both highest mRNA and gDNA quality and yield without any compromises. Material and Methods: As starting material for the mRNA purification from low-biomass input samples, the AdnaTest EMT 2/StemCell Select/Detect procedure was used. The procedure has already been tested widely to get highest yields of mRNA from as few as 2 cells spiked into 5 mL of blood. Cells captured with immunomagnetic beads as provided in the AdnaTest CTC-Select procedure were lysed using the AdnaTest Lysis/Binding Buffer and mRNA was purified using Oligo(dT)25 beads. After mRNA depletion cell lysates still contained gDNA, which can be isolated using magnetic affinity binding beads in an optimized workflow. mRNA recovery was demonstrated by reverse transcription and subsequent endpoint PCR. gDNA was analyzed using PCR and pyrosequencing as downstream applications. Results: It has been shown that the optimized workflow combination allowed the isolation of mRNA and gDNA from as few as 5 tumor cells spiked into 5 mL of blood. Tumor cell-related gene expression profiles can be analyzed after reverse transcription of mRNA and multiplex PCR. In parallel, the recovered gDNA was successfully used in PCR assays as well as for detection of known gene variants by means of pyrosequencing. Furthermore, gel electrophoretic analysis of the gDNA eluates showed good gDNA integrity with mean molecular weight values of >60 kbp. Citation Format: Jens van de Flierdt, Markus Sprenger-Haussels, Michael Otte, Siegfried Hauch. An optimized method of multimodal mRNA and gDNA isolation from low biomass input [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B44.