Life Science
by Donna McDade Walker
Determining the Quantity, Integrity, and Molecular Weight Range of Genomic DNA Derived From FFPE Samples
such facilities as biorepositories and genomic core laboratories. Formalin fixation and paraffin embedding (FFPE) is a standard preservation method for the long-term storage of sample tissue, and these samples are an important resource for the retrospective molecular study of clinical condi- tions. FFPE samples maintain tissue structure for histological examination; however, the nucleic acids contained within the samples can be of very poor quality.
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For genomic studies, high DNA yield and integrity are required to ensure reliable and reproducible assay results, but the fixation and embedding process, in conjunction with the storage duration, can lead to fragmenta- tion of genomic DNA. The quality assessment of FFPE blocks can therefore be challenging, since the quality of the extracted material can differ from block to block, and is dependent on the success of the extraction method. To ensure that genomic DNA is of the quality required, each sample must be screened to determine its suitability for downstream applications before committing time, money, and resources.
Traditional QC methods for FFPE genomic DNA The quality assessment of genomic DNA is usually performed by several
methods. In contrast to subsequent steps in the aCGH and NGS work flow, in which shorter fragment analysis can be performed by automated systems, the integrity assessment of the genomic DNA starting material is normally performed by agarose slab gel. This is a slow, labor-intensive, and manual process that can take several hours, and is in stark contrast to a largely automated, overall experimental work flow. In fact, to avoid this laborious, yet important, first step, some researchers choose to skip the initial QC in the hope that they can assess the quality further downstream. In addition, agarose slab gels are semiquantitative at best, meaning that an additional method is required to measure the genomic DNA concentration, which is usually performed by spectrophotometry or fluorometry.
Single-step method for genomic DNA QC To address the issue of a multiple-step manual process, Agilent Technologies (Edinburgh, U.K.) developed the Genomic DNA ScreenTape in conjunction with the 2200 TapeStation instrument. The Genomic DNA ScreenTape is a prepackaged, ready-to-use consumable device that con- tains a gel matrix and buffer specifically designed to reproducibly analyze large molecular-weight genomic DNA.
ssessing the quality and quantity of genomic DNA is a crucial step before next-generation sequencing (NGS) and DNA microarrays— for example, array comparative genomic hybridization (aCGH)—in
The 2200 TapeStation is an automated system that loads, electrophoreses, images, and analyzes results at the touch of a button. The digital analysis file in the 2200 TapeStation software provides a gel image for the sample integrity assessment, while also detailing the quantification and size of genomic DNA in less than 2 min per sample.
System technology: ScreenTape The ScreenTape is a credit-card-sized device made of three layers, each of which is composed of proprietary polymers that were carefully selected for their combinations of mechanical, thermal, optical, and biocompat- ible properties:
• The bioprocessing layer is formed to contain the 16 individual separation channels and buffer chambers
• The electrode layer contains the electrodes directly printed onto the polymer
• The protective carrier layer maintains the ScreenTape shape and catches any buffer overflow when in the TapeStation instrument (see Figure 1).
These three layers adhere together to form a ready-to-use, disposable device comprising 16 separate channels, each filled with a gel matrix and running buffer. By forming individual lanes, users can run the exact number of samples they wish; there is no need to batch samples. If less than 16 samples are analyzed, the ScreenTape can be stored in the refrigerator for an additional two weeks after initial use, so the remain- ing lanes can be run at a later date. This feature allows full scaleability and precise budgeting of cost-per-sample, since only the sample lanes required are used.
Figure 1 – The credit-card-sized, ready-to-use Genomic DNA ScreenTape consumable device is comprised of three separate polymer layers, specifi- cally designed for the separation of biomolecules through a gel matrix in 16 individually packaged channels.
AMERICAN LABORATORY • 12 • SEPTEMBER 2013
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