Determining the Quantity, Integrity, and Molecular Weight Range of Genomic DNA Derived From FFPE Samples

Assessing 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 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 conditions. FFPE samples maintain tissue structure for histological examination; however, the nucleic acids contained within the samples can be of very poor quality.

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 fragmentation 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 contains a gel matrix and buffer specifically designed to reproducibly analyze large molecular-weight genomic DNA.

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 biocompatible 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).
Figure 1 – The credit-card-sized, ready-to-use Genomic DNA ScreenTape consumable device is comprised of three separate polymer layers, specifically designed for the separation of biomolecules through a gel matrix in 16 individually packaged channels.

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 remaining 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.

In addition to the significant time-savings resulting from the use of prepackaged reagents, sample preparation is very simple. The Genomic DNA ScreenTape requires that only 1 μL of genomic DNA is vortex-mixed with 10 μL sample buffer; then the sample is ready to be placed into the 2200 TapeStation instrument for analysis.

System technology: 2200 TapeStation instrument

The TapeStation instrument was specifically engineered to analyze all ScreenTape types (for the analysis of DNA, RNA, and protein), and the system is designed so that no instrument priming is required. Not only does this save precious time, but it also allows the swift exchange between sample types. For instance, changing from the analysis of genomic DNA to smaller DNA fragments further downstream the experimental work flow requires only that the ScreenTape type be changed. This involves removing the tape from its sealed foil package and placing it into the instrument. The instrument contains a barcode reader that automatically reads the barcode contained on each ScreenTape. This provides instant recognition of the ScreenTape type inserted as well as tracking of any previously used lanes.

Figure 2 – The 2200 TapeStation instrument loads samples from tubes or a 96-well plate onto the ScreenTape, then electrophoreses, images, and analyzes the results in less than 2 min per sample.

The instrument can also automatically detect the type of sample block inserted—either tube strips or a 96-well plate. Once the samples, loading tips, and ScreenTape have been inserted, the 2200 TapeStation instrument is ready to go. To initiate the analysis process, the samples are selected and the “start” button is pressed. Inside the TapeStation, the instrument picks up a loading tip and simultaneously pierces the top of the lane it is going to load (see Figure 2). It then picks up the sample using the loading tip and places it on top of the gel matrix contained in the ScreenTape lane. After all the samples selected have been loaded onto the ScreenTape, the electrical probes in the instrument engage with the electrode pads at the top and bottom of each lane. A voltage is then applied across each lane, including a correction for gel temperature as measured by the TapeStation instrument.

When electrophoresis is complete, each lane is imaged and processed to calculate the parameters required for the specific ScreenTape assay, as determined by the barcode. Results are presented within approximately 1–2 min per sample.

System technology: 2200 TapeStation software

Figure 3 is a screenshot of the results file automatically launched for the Genomic DNA ScreenTape when the run is complete and the gel image has been fully analyzed. The default screen provides a gel image of all the selected samples, and the optional sample well is shown for visualization of any sample retained at the gel interface. Lane 1 depicts the genomic DNA ladder, which is supplied as part of the reagent kit; lanes 2–15 show a range of FFPE samples from various sources. Each lane contains a marker at 100 bp, which allows even small degradation products to be identified and acts as an internal standard for sizing and quantification.

Figure 3 – Screenshot of the automatically launched analysis file for Genomic DNA ScreenTape by the 2200 TapeStation software. The gel image shows DNA-extracted FFPE samples from different tissues: lane 1, ladder; lanes 2 and 3, prostate; lanes 4–7, pancreas; lanes 8 and 9, endocervix; lanes 10 and 11, lung; lanes 12 and 13, kidney; lanes 14 and 15, bladder. (Data courtesy of Dr. Toumy Guettouche, Hussman Institute for Human Genomics, Genomics Core, University of Miami, FL.)

The data table details, as default, the start and end of the sample in base pairs as well as the molecular weight size of the peak maxima. Concentration details in ng/μL are also provided with an accurate determination of the double-stranded DNA in each sample. Additionally, the software will flag whether the sample analyzed was outside the recommended concentration range (10 ng/μL–100 ng/μL).

Figure 4 – a) Gel image export with optional sample well of DNA-extracted FFPE samples from breast tissue (courtesy of Dr. Toumy Guettouche). b) Gel image scaled to molecular weight range of human genomic DNA extracted from FFPE blocks (courtesy of Agilent Technologies, Uppsala, Sweden).
Figure 5 – The sample comparison function of the 2200 TapeStation software allows a direct comparison of multiple samples from multiple files to ensure consistent sample quality over time.

The export function of the 2200 TapeStation software permits access to the gel images in .jpeg format. Figure 4 shows two exported gel images from breast tissue FFPE samples (Figure 4a) and human DNA extracted from FFPE blocks (Figure 4b). This gel image is superior to those generated by agarose gel and allows a visual check of the FFPE sample molecular weight and integrity. In contrast to an agarose gel, however, the image was generated in less than 2 min per sample with only a few minutes’ hands-on time and no exposure to harmful chemicals. In addition, the results generated are extremely reproducible due to the prepackaged and therefore standardized separation gel and buffers contained in the ScreenTape channels. In addition to the gel image, the software provides sample electropherograms and the ability to compare profiles from multiple samples in multiple files (Figure 5). This feature is invaluable for comparing successful and unsuccessful samples from previous NGS or aCGH experiments to current sample preparations and extractions.

Conclusion

The 2200 TapeStation system is a novel method for determining the concentration, integrity, and molecular weight size of FFPE genomic DNA in one step using only 1 μL of starting material. The technology automates a manual, laborious process and reduces hands-on time and overall time to result from ~2 hr to less than 2 min per sample. The system analyzes genomic DNA in the concentration range of 10 ng/μL–100 ng/μL and has a sensitivity of 0.5 ng/μL. The Genomic DNA ScreenTape, combined with the 2200 TapeStation, offers speed, scaleability, and simplicity to the genomics researcher performing NGS and aCGH assays with FFPE samples.

Donna McDade Walker, Ph.D., is Product Marketing & Support Manager, Agilent Technologies UK Limited, 5 Lochside Ave., Edinburgh Park, Edinburgh EH12 9DJ, U.K.; tel: +44 (0) 131 452 0715; e-mail: donna.mcdadewalker@agilent.com.

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