Abstract
BackgroundWith increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation.Results864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage.ConclusionsThe technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage.
Highlights
With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand
Efficient methods for biomolecule extractions from many tissue samples simultaneously are key components of the future workflow in molecular profiling of tumors, in particular in cancer biobanking, research and diagnostics
Automated serial extraction of nucleic acids We recently described a novel process for serial DNA and RNA extraction employing silica beads with differential nucleic acid binding affinities [5]
Summary
Automated biomolecule extraction from tissues The serial DNA/RNA extraction method described in [5] was successfully implemented in a fully automated fashion on a Tecan Freedom Evo workstation for parallel extraction of 96 samples or 1–96 samples using 8-channel liquid handling. We. Conventional and next-generation sequencing analyses of genomic DNA The extracted DNA was first used in PCR-based short tandem repeat (STR) analysis to compare genomic loci between tumor/normal matched samples to ensure that they were correctly paired. 400 – 800 ng DNA (as measured by Qubit) from 96 pairs of matched tumor and normal colorectal tissue (192 samples in total) was used in a Haloplex target. We have developed a walk-away automation solution to process fresh-frozen tissue specimens to high quality biomolecules ready for use in cancer research and diagnostics This novel technology enables the simultaneous processing of many different types of tissue samples in the pathology biobank workflow with a time and cost reduction for the user.
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