November 15, 2013 (Vol. 33, No. 20)

Approach Mixes UV/VIS with Spectral Content Profiling for Yield and Quality Assessment

Molecular genetic assays strongly rely on well-performed nucleic acid isolation as contamination with constituents from the sample or extraction reagents may interfere with downstream processing prior to functional nucleic acid analysis.

Traditionally DNA or RNA extracts are quantified by A260 spectrophotometric absorbance. Ratios A260/A230 and A260/A280 are the common methods for purity assessment; however, they only provide a rough estimation as they are influenced by sample characteristics like pH and salts and don’t provide any information related to RNA contamination in DNA samples. This is contradictory to the required quality control needed for complex genetic tests like qPCR or sequencing.

In addition, labs often turn to fluorescent dye-based assays like Picogreen or Qubit assays (Life Technologies) for specific quantification of the extracted DNA sample. However, the strength of these assays in terms of specificity comes with a significant price in respect to convenience because they require additional dispensing and mixing steps and the need to run standard curves in parallel.

Overall, a one-step approach characterizing the composition of a sample in terms of DNA or RNA yield and identification of contaminants would lead to improved process time and overall quality of the complete analytical workflow.

Trinean provides a solution that overcomes the typical problems of microvolume DNA/RNA QC: Both its DropSense96 and Xpose platforms are designed around microfluidic sample carriers for microvolume sample preservation and standardized read-out combined with innovative profiling of UV/VIS spectral curves for detailed sample content analysis beyond the possibilities of commonly used quantification methods.

Accelerating Quantification

Miniaturized DNA and RNA quantification has become a standard as it allows for the measurement of a broad concentration range and offers higher reproducibility by avoiding sample dilution steps. Trinean has further optimized this approach in terms of measurement speed, automation capability, and limiting repetitive manipulations by developing a microfluidic sample holder containing 16 or 96 sample positions.

As shown in Figure 1, each sample read-out structure consists of a pipetting inlet, capillary storage, microcuvettes, and an aspiration outlet with a waste trap. The pipetting inlets are conical recipients at a 9 mm pitch that allow easy pipetting with manual single or eight-channel pipettes as well as by liquid-handling robots.

When a two microliter sample is applied, the sample is spontaneously taken up into the storage area by capillary force. Once inside the holding pattern, the sample is protected from evaporation, without measurable change in readout for about two hours. The sample is also protected from cross-contamination and will not leave the capillary, preventing contamination.

This allows the preparation and loading of all samples at the workbench or in a liquid-handling robot and takes only the chip as sample container to the reader in a convenient and safe way. Once inside the reader, each sample will be transferred by aspiration to the microcuvette(s) with path lengths of 0.1, 0.5, or 0.7 mm depending on the selected microfluidic chip type.

The design and uniformity of the microcuvettes allows for the measurement of very precise UV/Vis spectra, which is needed for the spectral content profiling analytics. Within a single chip a large dynamic range from 0.03 OD up to 200 OD (corrected to 1 cm path length) is available. The measuring speed of both the Xpose and DropSense96 reader is approximately 4 seconds/sample, leading to a reading time of a 96-well microfluidic plate of 5 minutes.

Figure 1. Trinean microfluidic sample carriers with unique design including (1) microcuvettes for stable sample read-out; (2) input wells for manual or robotic sample dispensing; (3) storage meander with sample preservation.

Spectral Content Profiling

Compared to traditional A260 DNA or RNA quantification, dye-based measures are preferred for accurate quantification given the expected range of sample purity. To overcome the challenges inherent with these laborious dye-based assays, an innovative analytical tool was developed by Trinean based on detailed analysis of measured UV/VIS spectral shapes to determine the actual fraction of the DNA or RNA extract present, while accounting for interfering impurities.

New analysis software for spectral content profiling uses a mathematical model to unravel the measured spectra into the relevant molecular profiles that contribute to the absorption. This approach requires minimal handling steps while offering high specific DNA fraction quantification and assessment of the amount of contributing quantities of co-purified substances including sample turbidity, RNA, phenol, and others present in the sample.

To demonstrate the principle of spectral content profiling, a comparative analysis is performed using a commercial available genomic DNA (Sigma) spiked with an equal volume of either storage buffer, calf liver RNA (Promega), or phenol (Sigma). All three samples were measured in quadruplicate on a microvolume photospectrometer for A260 quantification, fluorometric-based DNA quantification, and the Trinean Xpose with Slide-40 microfluidic carriers. On the Xpose, the spectral content profiling app for mammalian DNA was selected.

Results in Figure 2 show that microvolume photospectrometry (MVS) overestimates DNA concentrations when contaminated, while DNA quantification by fluorometric assay (FMA) and the Xpose seem unaffected. Furthermore, purity analysis by the A260/A280 and A260/A230 ratios indicates “pure” DNA in all samples with values of 1.8–1.85 and 2.1–2.3.

Figure 2. Proof-of-principle of spectral content profiling: (A) Results as analyzed by the Xpose. (B) Overview of the DNA quantification results by single-channel micro­volume spectrophotometer (product A) using A260 quantifica­tion, Picogreen DNA assay by fluorometer (product B), and spectral content profiling by Xpose. Averages and standard deviations (STDEV) of fourfold measurement are shown.

In comparison, spectral content analysis by the Xpose does identify impurities indicated as A260 contributions when present, while FMA doesn’t give information on sample contaminants.

The DNA quantification specificity of the spectral content profiling was assessed with the courtesy of a molecular genetic lab using the Trinean DropSense96. Here, 64 DNA extractions from lymphoblastoid cell lines using a salting-out procedure were measured on an eight-channel microvolume spectrophotometer, a DropSense96 with spectral content profiling, and compared to the FMA considered as a DNA-specific method.

Figure 3A shows an example of a spectral profiling analysis on one random sample showing mainly contamination of RNA and EDTA. Figure 3B shows an overview of the MVS A260 results and DropSense96 spectral profiling results compared to the FMA data over a wide DNA concentration range between 30 and 300 ng/µL. This comparison demonstrates that the spectral content profiling results are similar to the FMA data (slope = 1.03), while the A260 results overestimate the actual DNA content (slope = 1.49), probably because of RNA contamination, as illustrated in Figure 2A.

In conclusion, the Trinean microvolume readers Xpose and DropSense96 in combination with spectral content profiling offer a fast, one-stop solution for accurate DNA and RNA quality and quantity assessment.

Figure 3. DNA quantification specificity comparison: (A) Spectral content profiling screenshot illustrating the reference profiles used and a single sample content analysis. (B) Comparative graph between fluorometric-based DNA quantification and microvolume spectrophotometry A260 results (grey) or DropSense96 (DS96) spectral content profiling results. Trend lines with their slopes are shown to highlight the overall conformity with fluorometric results.

Kris Ver Donck ([email protected]) is head of application development at Trinean.

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