Double-stranded DNA (dsDNA) must be quantified and normalized before it can be used in very many applications in genomics. These include PCR and next-generation DNA sequencing. Advanced fluorescence-based techniques which employ dsDNA-binding dyes that do not bind significantly to single-stranded DNA has made it possible to quantify minute volumes of dsDNA selectively, going a step beyond absorbance methods.
The Fluent Laboratory Automation Solution and FluentControl™ software are designed to speed up and automate this process. This article describes the advantages of this system in terms of simplified workflows, obviate manual analyses and provide increased scope for adapting the workflow for normalization of DNA by the use of Smart Commands in this software.
- Store Well Concentration stores the concentration reported for each sample after accounting for potential dilution steps
- Normalization commands makes use of recorded data to create a script by which the required volume and concentration of each sample can be achieved, whether in the same plate or another one
Once the Fluent platform is fitted with a microplate reader, the Quant-iT PicoGreen Kit runs itself through the process. It allows up to 96 samples to be diluted and quantified in triplicate, using a 384-well plate, and generating a 4-point standard curve. The time taken for the whole run is only 80 minutes, of which 55 minutes goes in setting up the quantification plate, 10 minutes for reading and 15 minutes for normalizing the samples. This time includes the automatic dilution of the sample based on the highest concentration that is estimated in the plate containing samples.
The intuitive TouchTools™ touchscreen interface helps the user understand how to set up the instrument and navigate the selection options with ease, as well as thereby making sure the experiment complies with SOPs and enhancing the security of the process from variable factors.
Figure 1: Schematic workflow of the Fluent quantification and normalization protocol. (1) Samples prediluted (not shown) and pipetted into the quantification plate. (2) Concentration of each sample measured and stored. (3) Samples normalized in a new microplate.
Materials and methods
This protocol for dsDNA quantification with the Quant-iT™ PicoGreen® dsDNA Assay Kit was designed on the Fluent workstation fitted with an eight-channel Air Flexible Channel Arm (FCA) using filtered single-use tips, a Robotic Gripper Arm and an Infinite® F Nano+ setting of the Infinite 200 PRO reader placed under the worktable, as seen in Figure 2. The run was controlled by the Store Well Concentration and Normalization Smart Commands of the FluentControl™ software.
Figure 2: Deck layout of the Fluent workstation for quantification and normalization.
The dsDNA was quantified using the Quant-iT PicoGreen Kit as per the manufacturer’s instructions. The concentration of one single sample of K562 genomic DNA from Promega was assayed eight times in five runs to evaluate the degree of reproducibility within and between runs. This was followed by the generation of a standard curve ranging from 5 to 2000 ng per μl of dsDNA.
It is easy to use the Fluent station because of the way the touchscreen interface provides instructions on screen to guide the operator through the set-up and prompts when any parameter is to be defined, such as:
- The number of samples (between 1 and 96)
- The number of replicates (1-3)
- Whether a new standard curve is to be generated or stored data is to be analyzed
- Whether dilution of the sample should be performed prior to quantification
- The final volume to be achieved with normalized samples
- The final concentration of samples following normalization
The total time for the complete workflow including quantification and normalization was about 80 minutes, which includes the time for setting up an intermediate dilution plate and taking fluorescence readings in triplicate.
The Normalization Smart Command is capable of being set to handle samples which fall outside the prespecified limits of concentration either automatically or after requesting an action to be defined by the user.
Analysis and results
Each of the five runs in this experiment was analyzed by calculating the concentration of each replicate by linear regression as per the manufacturer’s manual (Table 1). The results obtained showed high consistency within runs and between the five runs, with CV < 5.5%. Figure 4 is the representative standard curve for this assay.
Table 1: Consistency of standard curve preparation based on five runs of eight replicates for a simple sample.
Figure 4: Representative dsDNA standard curve using the Quant-iT PicoGreen dsDNA Assay Kit.
The results were also shown not to undergo change when 96 samples were used, 48 at high concentration and 48 at low, arranged as if on a checkerboard. The samples checked from various parts of the plate also showed consistency of results, which is evidence of lack of cross-contamination as shown in Figure 6.
Figure 5: Quantification results for the assessment of cross-contamination using the automated method (Darker shading = average concentration 84.7 ng/μl; Lighter shading = average concentration 34.1 ng/μl).
Figure 6: Quantitation results for samples normalized to 25 ng/μl using the automated method. The observed average concentration was 25.9 ng/μl, with a CV of 5.5%.
The normalization of quantified samples was then carried out to achieve a final concentration of 25 ng per µl. Repeat quantification was done following this step, to make sure the Normalization Smart Command was reliable. On average, the normalized samples showed a concentration of 25.9 ng/μl with a CV of 5.5%.
The data obtained proves the excellent efficiency and reproducibility of the Quant-iT PicoGreen dsDNA Assay Kit used on the Fluent platform when used to quantify and normalize dsDNA. The Fluent workstation offers splendid adaptability with easy use through the user-friendly TouchTools™ UI.
The range of options that can be set through this interface requires minimal training but allows a wide variety of workflows to be run, including QC after DNA sample purification and NGS library-quality sample preparation. The use of an MCA pipette with 96 or 384 channels is optional if still faster processing is required.
- Quant-iT PicoGreen dsDNA Reagent and Kits manual. MP 07581. Thermo Fisher Scientific.
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