Ins and outs of nucleic acid quantification and the DropSense solution towards standardization Thomas Martens, Kim Plasman, Tine Duthoit & Tony Montoye Trinean NV, Dulle Grietlaan 17/3, 9050 Gentbrugge, Belgium
Introduction Failure of many molecular assays can be attributed to the quality of the nucleic acids used. Therefore, it is of utmost importance to thoroughly investigate both the quantity and purity of nucleic acids before proceeding to downstream assays. Most commonly-used technologies include UV/VIS droplet spectroscopy (i.e., NanoDrop) or fluorescence-based assays (i.e., Qubit). These however suffer from some inherent drawbacks like labor-intensity and low accuracy. In an attempt to increase specificity, preserving ease of use and processing speed, UV/VIS droplet spectroscopy and an intelligent algorithm (DropSense16 + cDrop) were combined, allowing more accurate nucleic acid quantification. Here, we use experimental data using different types of nucleic acids to demonstrate the advantages and shortcomings of each technology and propose ways to standardize nucleic acid QC, ultimately resulting in a higher success-rate of downstream assays.
DNA/RNA quantification options
DropSense16 properties Load 2 µl samples on a microfluidic slide. No evaporation or cross contamination. Simply insert the slide and select an application. 16 samples are measured in about 2 minutes.
Blood Saliva Tissue Cell lines DNA/RNA extraction
Fluorescent assay Quant-iT/Qubit
DropSense16 + cDrop
DropSense16 DNA/RNA quantification
NanoDrop A260
Both fluorescent assays as well as NanoDrop measurements are considered gold standards in genomic labs. Qubit and other fluorescent assays are used for specific quantification of DNA and RNA samples while NanoDrop is used for quick quantification and purity analysis using the A260/A280 and A260/A230 ratios. Here, we compare both methods with the DropSense dye-free technology using classic UV/VIS absorbance combined with an advanced algorithm included in the cDrop software, allowing dissection of the measured UV/VIS profile to identify contaminants in the sample and correct the DNA or RNA concentration accordingly.
Factors influencing the selection of a quantification method 1. Purity of the isolated DNA or RNA sample
Interlab differences were investigated measuring an identical set of samples both at Trinean and at a US based collaborating lab. Samples 2-4 and sample 6 contain DNA purified from whole blood with Qiagen’s EZ1 extraction kit whereas samples 1 and 5 hold SPRI purified calf thymus DNA (Thermo Fisher Scientific). Samples were analysed using Trinean’s DropSense (DS) platform with and without the ‘DNA mammalian’ cDrop application in addition to Qubit analyses. Bar charts below clearly indicate that, while DS A260 and DS + cDrop analyses are less subject to variations (A), Qubit measurements tend to vary more (B). To highlight the importance of the choice of reference for Qubit analyses, in both labs, Qubit measurements were performed using a genomic DNA (i.e., SPRI DNA) or Qubit standard reference (C,D). Qubit measurements with the Qubit standard as reference underestimate genomic standard reference Qubit results 15% on average. A. DNA concentration (ng/μl)
DNA samples were provided by cooperating labs amounting to a total of 836 samples from different mammalian sources (blood, tissue, saliva, cell lines) using 13 different extraction kits, ranging from bead-based to columnbased and salting-out extractions. DNA concentration of all samples was determined by 2 direct and one indirect, fluorescence-based quantification method. The direct quantification methods include droplet A260 spectrophotometry and Trinean’s cDrop ‘DNA mammalian’ app on the DropSense16; the indirect method of choice is Quant-iT PicoGreen (Thermo Fisher Scientific). The plot chart below demonstrates that the results of the ‘DNA mammalian’ app (blue crosses) nicely correlate to values obtained with Quant-iT PicoGreen (y = 1.04x). Classic A260 spectrophotometry (gray crosses) however, tends to overestimate these (y = 1.24x) due to the presence of UV-absorbing impurities.
3. Complexity of the quantification method
C.
Interlab difference DS +/- cDrop
250
250
200
200
Trinean DS A260 150
150
100
100
50
50
US lab DS A260 Trinean DS + cDrop US lab DS + cDrop Trinean qubit std
0 S1
B. DNA concentration (ng/μl)
Qubit @ Trinean
S2
S3
S4
S5
0
S6
S1
D.
Interlab difference Qubit
250
250
200
200
150
150
100
100
50
50
0
0 S1
S2
S3
S4
S2
S5
S6
S3
S4
S5
US lab qubit std
S6
Trinean genomic std
Qubit @ US lab
US lab genomic std
S1
S2
S3
S4
S5
S6
2. Size of the DNA or RNA sample
4. Time/cost of the quantification method
A commercial phage lambda DNA sample (Thermo Fisher Scientific) was acquired and mechanically sheared during different numbers of cycles (1 minute) to obtain fragmented DNA of different sizes. These were subsequently quantified using UV/VIS (A260) and Quant-iT PicoGreen. The results demonstrate that the fluorescent assay tends to underestimate the amount of DNA up to >40% with reducing fragment lengths. Direct absorbance techniques like NanoDrop and DropSense16 are not influenced by fragment length. This observation is confirmed in the literature (refs 1,2).
A direct comparison of the costs and labor involved in DNA quantification by NanoDrop, Qubit, and DropSense16 is reported in the table below. Although Qubit is the less expensive device, the cost of its fluorescent dye and long sample processing time of over 5 minutes lead to a cost of 2.5$/sample. Both UV/VIS based systems have significantly lower costs of about 0.6-0.9$/sample. The DropSense16 has a slightly larger assay cost, due to its disposable microfluidic slide but as this allows higher sample processing speed; labor time and cost are significantly lowered. Over a full year use, significant cost savings can be achieved selecting an UV/VIS reader for mainstream DNA and RNA quantification. NanoDrop
Qubit
DropSense16
15 samples/day 30 samples/day
15 samples/day 30 samples/day
15 samples/day 30 samples/day
Assay cost Dye cost / sample Accesory cost (tips, wipes, ...) / sample Read-out vessel (tube, plate, slide) cost / sample
0.00 0.08 0.00
0.00 0.08 0.00
Total assay cost / sample
0.08
0.08
0.50 0.16 0.52 0.23
0.50 0.16 0.26 0.11
$
0.52 0.12 0.14
0.52 0.12 0.14
0.78
0.78
5.30 1.71 0.06 0.00
5.30 1.71 0.03 0.00
$
0.00 0.04 0.31
0.00 0.04 0.31
0.35
0.35
0.20 0.06 0.56 0.00
0.20 0.06 0.28 0.00
$
Labor + reader costs Prep + read time per sample (minutes, based on ref. 3) Labor cost / sample (labor cost based on ref. 4) Cap Equip amortized / sample (list pricing, 7 years ammortized) Yearly maintenance visit / sample
DNA_8’
DNA_6’
DNA_4’
DNA_3’
DNA_2’
DNA_1’
DNA_ctrl
(visit required)
(User Calibration)
(User Calibration)
Total labor + reader cost / sample
0.91
0.53
$
1.77
1.74
$
0.62
0.34
$
Total cost / sample Total cost / year Yearly cost comparison (% to Qubit) Yearly cost comparison ($ to Qubit)
0.99 3,270.25 -61.08% -5133.00
0.61 4,032.50 -75.72% -12576.00
$
2.55 8,403.25 0.00% 0.00
2.52 16,608.50 0.00% 0.00
$
0.97 3,215.50 -61.74% -5187.75
0.69 4,583.00 -72.41% -12025.50
$
$ $
$ $
$ $
References (1) Sedlackova T, Repiska G, Celec P, Szemes T, Minarik G (2013) Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biol Proced Online 15: 5 (2) Shokere LA, Holden MJ, Jenkins GR (2009) Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA. Food Control 20: 391-401 (3) Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpelli G, Lawlor RT, Scarpa A (2013) DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples. PLoS One 8: e62692 (4) Average salary of a lab technician in Biotechnology in the US. (http://www.indeed.com/salary?q1=lab+technician+biotech&l1)
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