Abstract 1965: Multimodal analysis of circulating cell-free RNA (ccfRNA), cell-free DNA (ccfDNA) and genomic DNA (gDNA) from blood samples collected in PAXgene Blood ccfDNA Tubes
Authors: Ullius Andrea , Provencher Eric , Voss Thorsten 
Published: September 2020

Abstract
INTRODUCTION
While circulating cell-free DNA (ccfDNA) from blood is widely used as an analyte in liquid biopsy cancer research applications, circulating cell-free RNA (ccfRNA) has only recently gained relevance for biomarker studies. Combined insights from both analytes promise to increase the understanding of molecular processes in tumors. However, challenges in the preanalytical workflow, such as choosing blood collection tubes and extraction methods suitable for multimodal analysis must still be overcome. We investigated multimodal extraction and analyses of ccfRNA, ccfDNA and gDNA from one blood sample collected using the PAXgene® Blood ccfDNA Tube*.
METHODS
Paired whole blood samples were collected from healthy consented donors into PAXgene Blood ccfDNA Tubes (PreAnalytiX®), BD Vacutainer® K2EDTA tubes (BD), Cell-Free DNA BCT® (Streck®), RNA Complete BCT™ (Streck) and LBgard® Blood Tubes (Biomatrica®). Plasma was generated by double centrifugation immediately after blood collection or after storage. Cell-free nucleic acids were extracted manually using the QIAamp® Circulating Nucleic Acid Kit (QIAGEN), the exoRNeasy Serum/Plasma Kit (QIAGEN®) and the miRNeasy Serum/Plasma Kit (QIAGEN). gDNA was extracted from the residual cellular fraction using the QIAamp DNA Blood Mini Kit (QIAGEN). QIAGEN qPCR assays were used to analyze ccfRNA. An internally validated qPCR assay was used for analysis of ccfDNA.
RESULTS
Quantitative PCR analysis of extracted ccfRNA revealed similar yields of miRNA, mRNA and ccfDNA targets from PAXgene and EDTA plasma. RNA detection sensitivity was lower for blood collected and stored in tubes containing formaldehyde-releasing formulations (Streck and Biomatrica) as indicated by higher CT values. Likewise, for gDNA, yield and purity were reduced by storage in Streck and Biomatrica tubes compared to blood stored in PAXgene tubes. Besides differences in yield, extraction of ccfRNA from plasma using the three tested isolation kits showed high sample reproducibility and precision for all collection tubes.
CONCLUSION
This study demonstrates the non-crosslinking technology of the PAXgene Blood ccfDNA Tubes enables the isolation and analyses of cell-free miRNA, mRNA, ccfDNA and cellular gDNA. These results are also in line with recent data reported indicating successful CTC enrichment and detection of RNA from blood stored in PAXgene Blood ccfDNA tubes. Taken together, the studies support a multimodal use of the PAXgene Blood ccfDNA Tube for in liquid biopsy research. The three tested ccfRNA isolation kits - the QIAamp Circulating Nucleic Acid, the exoRNeasy Serum/Plasma Kit and the miRNeasy Serum/Plasma Kit - enable high-quality isolation of ccfRNA. Other blood collection tubes for ccfDNA stabilization showed impaired extraction and detection efficiency for several mRNA and miRNA targets.
*For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Andrea Ullius, Eric Provencher, Thorsten Voss. Multimodal analysis of circulating cell-free RNA (ccfRNA), cell-free DNA (ccfDNA) and genomic DNA (gDNA) from blood samples collected in PAXgene Blood ccfDNA Tubes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1965.