DNA and RNA: Why It’s All About Integrity
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In this interview, we spoke with Knut Wintergerst, PhD, General Manager of Biomolecular Systems and Solutions (BSS) and Diagnostics and Genomics Group (DGG) at Agilent to learn more about why DNA and RNA sample integrity is important to scientists and how it can be maintained and analyzed.
Molly Campbell (MC): Why is it important to ensure that RNA and DNA samples are of a high quality?
Knut Wintergerst (KW): To avoid a garbage-in garbage-out situation it is essential to work with the highest DNA and RNA sample quality possible. Poor sample quality will have a major impact on the results of any downstream experiment as quality compromises sample concentration, size distribution as well as degree of degradation or sample integrity. DNA and RNA quality can vary significantly and depends on multiple factors such as nature of the source material, extraction method, sample age and storage conditions. An example of this is with DNA and RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue, which can exhibit broad degrees of degradation, is dependent on treatment of the tissue during fixation, as well as age and storage temperature prior to fixation. DNA, and especially RNA molecules, are fragile and can undergo fast degradation if not handled with precaution and stored correctly.
Tight sample quality control (QC) at multiple stages of the workflow is critical. Downstream applications are typically lengthy and expensive, and in the case of next-generation sequencing (NGS) will generate massive amounts of data. A huge number of samples are stored in Biobanks and must be of the highest possible quality to ensure clinical cohort studies yield conclusive and correct results. It is in the interest of researchers to work with the best sample quality possible to avoid re-runs or biased or false data.
Agilent developed integrity numbers for genomic DNA as well as total RNA samples to provide a user independent, easy tool for nucleic acid integrity assessment. The numbers range from 1 (fully degraded) to 10 (fully intact). On the Agilent QC platforms, such as the 4150 TapeStation and 4200 TapeStation, the integrity assessment is combined with determination of size, concentration and molarity, giving end users a complete picture of the quality of their samples in just one run.
Labs usually set an integrity number as the lowest allowed limit for the samples to be used in their workflow. This integrity number threshold, which is specific for the sample material and extraction method, as well as downstream application, is determined in a user independent fashion. Only samples passing this limit are supposed to be used for any further downstream experimental step. This ensures experimental success and saves time and money.
Among the scientific community, guidelines have been established to include sample quality details for specific downstream experiments MIQE (qPCR), MIAME (microarray analysis) as well as MINSEQE (high-throughput sequencing), which reinforce the use of QC tools. Especially in clinical settings, QC has become mandatory, to ensure maximum fidelity of the data and experimental results.
MC: Please can you explain the background of the Agilent 4150 TapeStation's development?
KW: With the introduction of the 4200 TapeStation system in 2015, Agilent provided a nucleic acid sample QC solution that meets the need of mid-to high throughput customer. The system can run 96 well plates or sample tubes, depending on the workflow of the customer, which are typically analysed in about 90 minutes.
From smaller, low throughput labs there has been growing demand for a cost effective, easy to use low throughput instrument, which offers all advantages of the ScreenTape technology.
The 4200 TapeStation and 4150 TapeStation share the same analysis software, accessories, consumables and ScreenTape for DNA and RNA analysis, making transitioning between the two TapeStations seamless, with no training required. Throughput of QC becomes easily scalable for any lab, minimizing lab method re-validation requirements, which is important for clinical labs.
The compact footprint of the 4150 TapeStation saves valuable bench space in the lab, which is a high need especially in smaller labs.
MC: How does the Agilent 4150 TapeStation analyze the quality of RNA and DNA samples?
KW: Both TapeStation systems separate nucleic acids by means of gel electrophoresis. The electrophoresis occurs in a miniaturized, ready to use consumable called a ScreenTape device.
Prior to sample separation the operator mixes 1 or 2 ul of DNA and RNA sample with an application specific sample buffer that includes an intercalating, fluorescent dye used for detection. To support sample denaturation, RNA samples are briefly heat denatured. Prepared samples are provided in two 8x tube strips. After starting the run, the instrument dispenses the prepared samples in the ScreenTape device, lane by lane, thus avoiding any cross contamination. Each sample is analysed in an individual lane.
After electrophoretic separation of the sample, which takes 1-2 minutes, the DNA and RNA molecules are detected by the imaging system. Sample detection is based on light induced fluorescence. Data analysis is done automatically by the user friendly TapeStation software and results are presented in a gel image as well as electropherogram trace, in which fluorescence intensity is plotted against size.
Internal and external standards are used to calculate quantitative and sizing results that are provided in tabular format. Dedicated software algorithms were developed specifically for the analysis of genomic DNA and total RNA to provide integrity numbers, the DNA integrity (DIN) and RNA Integrity number equivalent (RINe), respectively. These numbers range from 10 (complete intact sample) to 1 (very much degraded sample) and allow an easy assessment of nucleic acid material from a variety of sources. Once users validated and defined individual integrity number thresholds, experimental success can be guaranteed.
MC: Please can you tell us more about your ScreenTape technology?
KW: The ScreenTape device is a credit card sized disposable that enables the separation of up to 16 samples in individual lanes, eliminating cross contamination risk. The ScreenTape device does not require any preparation. It comes ready to use.
Each sample lane includes a sieving polymer matrix, electrodes, as well as buffer chamber. The ScreenTape technology offers significant advantages like ease-of use, higher sensitivity, speed and resolution over classical slab gel electrophoresis. Depending on the assay, not more than 1-2 ul of sample is required and down to 5pg/ul DNA can be detected.
A large portfolio of different ScreenTape assays is available that address different customer needs in sizing range and level of sensitivity
ScreenTape devices are barcoded and are automatically detected by the instrument and assay specific settings like electrophoresis parameters, as well as data analysis settings, are automatically loaded. There is no need for running a specific sample number as a previously used ScreenTape device can be re-used within two weeks. This enables throughput independent costs per sample, which is an important feature for customers. Information on available lanes on a specific ScreenTape device is stored via the barcode within the software. Ease of switching ScreenTape assays is enabled as the user simply needs to only switch the ScreenTape device.
Agilent offers two instruments that are based on ScreenTape technology, the 4200 TapeStation system that enables the unattended analysis of up to 96 samples loaded from a well plate and the new 4150 TapeStation instrument, which analyses any sample number between 1 and 16. Full assay compatibility guarantees seamless switching between the instruments.
MC: What are the advantages of adopting the Agilent 4150 TapeStation technology over other available technologies in the market?
KW: The 4150 TapeStation system is the first instrument based on ScreenTape technology that specifically addresses the needs of price-sensitive, low-throughput customers having limited bench space in the lab. The ScreenTape technology offers significant advantages like higher sensitivity, speed and resolution over classical slab gel electrophoresis. In addition, it provides accurate and reproducible results, independent of the operator.
Besides this, the exceptional ease of use (all ScreenTape come pre-packaged and ready to use), as well as throughput independent cost-per sample, are unique features. For reliable, easy and reproducible nucleic acid sample quality control, Agilent offers integrity numbers for extracted genomic DNA (DNA Integrity number, DIN) as well as the RNA integrity number equivalent (RINe).
Low cost of ownership is guaranteed by few and easy maintenance steps that can be performed by customers, who also have access to Agilent worldwide, world class support infrastructure with trained service technicians and multiple service options.
Knut Wintergerst was speaking to Molly Campbell, Science Writer for Technology Networks.