Making Double-stranded DNA Quantification and Normalization easy and fast using Promega QuantiFluor® dyes with the Fluent® Laboratory Automation Solution from Tecan.
The field of genomics demands double-stranded (ds) DNA quantification and normalization to a high standard of reliability. While absorbance techniques have been used in classical genomics studies, many newer techniques have emerged over time, among which techniques based on the selective binding of fluorescent dyes to dsDNA (but not to single-stranded DNA or RNA) have become widespread because of its ability to detect minute amounts of nucleic acid.
The Fluent automated laboratory platform with FluentControl™ software has boosted the power of workflows in genomics besides enabling automatic operation. This article shows the use of FluentControl Smart Commands in this software to simplify and automate processing and calculations, while enhancing the ease of modification of the protocol to suit different workflows.
The Smart Commands include:
- Store Well Concentration: this command saves each sample’s concentration, with regard to potential dilution
- Normalization: this command makes use of recorded data to generate an automated script to fill each sample in the current or a new plate, at the required concentration and volume
The Fluent platform has an inbuilt microplate reader to enable increased walkaway time, as well as a higher capacity. It lets the operator dilute and quantify up to 96 samples in triplicate as well as the standards needed to generate an eight-point standard curve, using a microplate with 384-well format.
The whole workflow is completed in just 75 minutes, including 55 minutes to set up the quantification plate, 5 minutes to read it, and 15 minutes to normalize the samples. The final results include automatic dilution of the samples based on the maximum estimated concentration within the sample plate.
The platform also has an intuitive and simple-to-use interface called TouchTools™ which is a touchscreen that helps the user to easily understand how and when to select sample parameters such as:
- Sample number
- Sample replication
- Final concentration
- Target volumes
The interface also guides the operator through the set-up process so that the experiment complies with SOPs and the process is secured against unwanted variability, ensuring high consistency of results.
Materials and methods
In this experiment, a Fluent platform was used with an attached Flexible Channel Arm based on air displacement with eight channels, with filtered single-use tips, a Robotic Gripper Arm, and a multimode Infinite® M1000 reader set up as in Figure 2. The process run was controlled by the Store Well Concentration and Normalization Smart Commands on the FluentControl software.
The dsDNA was quantified with the QuantiFlor dsDNA system as per manufacturing instructions. A sample of K562 genomic DNA1 was run through the process five times and measured eight times during these runs, so as to evaluate the reproducibility of the results between runs and within a run. The results were analyzed and used to generate a standard curve ranging from 5 to 2,000 ng per µl of dsDNA.
The worktable was set up following the on-screen instructions of FluentControl software, and requested parameters were selected, some examples being shown in Figure 3:
- Sample number between 1 and 96
- Replicate number between 1 and 3
- Whether recorded data or a standard curve newly generated is to be used
- If required, to what ratio the sample is to be diluted pre-quantification
- Final normalized sample volume
- Final normalized volume concentrations
The full workflow was completed in 75 minutes including preparing an intermediate dilution plate as well as taking fluorescence readings in triplicate.
The Normalization Smart Command can be set to either ask for a user-specified command when the system comes across a sample out of predefined concentration ranges, or to handle such samples automatically.
Analysis and results
As Table 1 shows, each replicate was analyzed for concentration in each of the five runs using linear regression techniques in compliance with the QuantiFlor dsDNA manual. The experiment led to the obtaining of very similar results across all runs and within the same run, with a CV (<4.5%. One standard curve is shown in Figure 4 to represent those obtained by this system.
In addition, when quantification values were cross-checked using 48 samples at high-concentration and low-concentration respectively in a checkerboard layout, the results showed no significant variation in any part of the plate ruling out any cross-contamination, as shown in Figure 5.
Finally, the quantified samples from the checkerboard layout were subjected to normalization to achieve a final concentration of 2.5 ng per µl, and then quantification was repeated to make sure the Normalization Smart Command was producing repeatable results. This showed the normalized samples had a concentration of 2.6 ng/μl (CV of 6.9%) on average.
In light of the data obtained from this experiment, it is clear that using the Fluent platform with the QuantiFlor dsDNA system allows ease of operation, increased flexibility of workflow, and highly consistent results in quantification and normalization of dsDNA.
It may be modified to suit other kinds of workflows such as those involved in quality control of purified nucleic acid or preparation of samples for the NGS library, because it is simple to use the software commands to alter the parameters according to requirement, as well as the optional use of a Multiple Channel Arm for pipetting through either 96 or 384 channels as per need.
Produced from materials originally authored by Dr. Enrique Neumann, application scientist at Tecan.
- QuantiFluor dsDNA System Technical Manual. Document TM346. Promega Corporation.
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