Abstract 3634: Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method
Authors: Aleksandra Ras, Samantha Helm, Kevin Kelly, Vanessa Spotlow, Guruprasad Ananda, Gregory Tsongalis
Journal: Cancer Research (2016)

Abstract Introduction: The purification of genomic DNA (gDNA) and total RNA (RNA) from various specimen types using a single sample is currently in high demand in genomic settings due to the limitation of available biological material. There is an increased need for simultaneous isolation of nucleic acids in cancer genetics for use in downstream applications such as genotyping, microarrays or next generation sequencing. The Qiagen AllPrep® technique provides a comprehensive solution for extraction of gDNA and RNA from a single specimen. In this study, we tested the AllPrep® kits for concurrent isolation of gDNA and RNA from five different sample types. Materials and Methods: Genomic DNA and RNA were extracted simultaneously according to Qiagen AllPrep® manufacturer's instructions from five specimen types: bone marrow mononuclear cells (BMMC), cryopreserved cultured cell lines, whole blood, fresh frozen and FFPE samples. Quantity and quality of both nucleic acids were assessed by the NanoDrop 2000 and Qubit fluorometer 2.0. Purified gDNA from BMMC, fresh frozen, whole blood and FFPE samples was further evaluated by library preparation, targeted hybridization capture and sequencing on an Illumina NextSeq500. FastQ files were submitted to a Clinical Genome Analytics (CGA) pipeline for quality metrics analysis. Results: The AllPrep® technique resulted in effective isolation of high quality gDNA and RNA (Table1). All set wet lab and sequencing QC metrics were met for each sample type. Conclusion: We have successfully verified the AllPrep® method can be used for simultaneous extraction of both gDNA and RNA using the same input material without compromising the yield or purity of both final products. This simple procedure based on spin column technology not only gave the advantage of generating high quality results using limited specimen resources, but also decreased the hands on time by 3.5 hours and cost when compared to extraction done with two separate kits. Table 1.Represents average QC results and SD for each specimen type. **n = 8 for sequencing metricsgenomic DNAgenomic DNAtotal RNAtotal RNASample TypeOD260/280Total Yield (ng)OD260/280Total Yield (ng)Mean Target Coverage% DuplicationBMMC (n = 3)1.853033.31.9413855206.34%Whole Blood (n = 2)1.818936.51.881335566.96.47%Fresh Frozen (n = 2)1.69312752.0432375345.44.02%FFPE Specimen (n = 16)1.665133.41.9410524.1**748.2**6.10%Cultured Cell Lines (n = 5)1.8442981.9810489N/AN/ATotal Average (n = 28)1.736898.141.9510443624.75.92%Standard Deviation (SD).249180.90.110616.2228.30.013% Citation Format: Aleksandra Ras, Samantha Helm, Kevin Kelly, Vanessa Spotlow, Guruprasad Ananda, Gregory Tsongalis. Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3634.