Normalization of samples refers to the process of equalizing sample volume or concentration to a desired level prior to any downstream application. This is often considered best practice for high quality and reliable data. Common applications include molecular biology protocols such as DNA library preparation for next-generation sequencing, normalization of RNA concentration prior to cDNA synthesis or protein concentration normalization prior to enzymatic assay screening.

This technical note outlines a workflow where samples were normalized to a specified target concentration into a new plate using the Normalize element. The workflow was executed on three liquid handlers: a Gilson PIPETMAX 268, a Hamilton Microlab STAR and a Tecan Freedom EVO. When using the Normalize element, Synthace automatically calculates mixture volumes when target and stock concentrations are provided to prevent human-error.

This technical note has been used to validate liquid handling accuracy for the volumes of liquid (tartrazine) transferred for normalization, and provides a starting point for liquid handling optimization should it be required.

Within this workflow (Figure 1), eight samples (Sample 1-8) with various concentrations of tartrazine (65-100 µM) were normalized by concentration to two separate assay plates.

Figure 1: Normalize workflow in Synthace. This workflow describes the method for normalizing samples over three automation devices. Tartrazine samples were defined with the Define Liquids And Plates element with unnormalized samples selected and aliquot to two assay plates and the remaining samples normalized to the same assay plates using the Select, Aliquot and Normalize elements, respectively.

Unnormalized samples were aliquoted in triplicate to a final volume of 100 µl to two final assay plates providing absorbance readings for the initial unnormalized samples. All samples were then normalized in triplicate to 50 µM in a final volume of 100 µl to each of the assay plates using reverse osmosis (RO) water as diluent (Figure 2). This workflow is executed over three devices, namely a Gilson PIPETMAX 268, a Hamilton Microlab STAR and a Tecan Freedom EVO.

Figure 2. Execution Details Preview in Synthace. Users can simulate their workflows, preview and verify their experiments in silico prior to physical execution demonstrated here on the Hamilton Microlab STAR.

Labware information and estimated execution run times for each of the liquid handling devices is shown in Table 1. The workflow was run on all three devices on the same day. Tartrazine absorbance was measured using a BMG Labtech CLARIOstar microplate reader at 425 nm (Tartrazine 𝜆max) and baseline subtraction performed with absorbance at 620 nm. Data was processed and graphs were generated by a python script in Jupyter Notebook and can be accessed from the Downloads section at the end of this article.

Table 1: Liquid handling information

Independent measurements (Figure 3), and intra- and interplate variability (Figure 4) of unnormalized and normalized Tartrazine samples were calculated from the data collected for samples prepared across the three devices tested.

The average intraplate coefficient of variance (CV) after normalization was 0.77% (Glison PIPETMAX 268), 0.97% (Hamilton Microlab STAR) or 0.61% (Tecan Freedom EVO), while average interplate CV was 1.23% (Glison PIPETMAX 268), 0.45% (Hamilton Microlab STAR) and 0.46% (Tecan Freedom EVO).

Figure 3. Absorbance data for unnormalized and normalized samples across three devices. Corrected absorbance values of the original tartrazine samples (left panels) and the normalized samples (right panels) for (A) Gilson PIPETMAX 268, (B) Hamilton Microlab STAR and (C) Tecan Freedom EVO. Each data point represents an individual replicate.

Figure 4. Coefficient of variation (%CV) for each set of replicates, without and with normalization. %CV for each set of replicates, either without or with normalization, on each plate (A) intraplate variation, and across both plates, (B) interplate variation. Dashed horizontal lines represent Synthace internal validation standards for the % CV maximum thresholds.

The following file contains the workflow that was discussed in this technical note. To download the file, right-click the button, then select Save as.

After you download the file, complete the following steps.

The following file contains the raw data that was discussed in this technical note. To download the file, right-click a button, then select Save as.

If you want to process the raw data in the way that was discussed in this technical note, download the Jupyter Notebook or Python scripts. You must edit the scripts to point at the directory that contains the raw data.