High-Quality & Automated qPCR Sample Preparation

Often, gene expression analysis and diagnostic testing rely on real-time PCR (qPCR). This powerful technique quantifies the amount of DNA in the sample [1]. The amplified DNA is detected in real-time by the incorporated fluorescent dye, for example SYBR Green, or dye-labeled oligo primers, like TaqMan probes.

The sensitivity of the dyes at the level of the picogram renders the detection dynamic range of ≥6 fold. Inconsistent pipetting or cross-contamination with a minute amount of DNA can dramatically skew the data and the quantification of DNA because PCR products are amplified exponentially. This invalidates the final results. Therefore, qPCR experiments need to be incredibly accurate and need to have guaranteed reproducibility.

This is particularly challenging when they are done manually because pipetting techniques vary within and among operators. These differences cannot be easily rectified, and require actions to correct them. For example: the pipetting speed should not be too fast, especially for viscous solutions of the master mix, to avoid splashing the samples; the pipettes need to be held at vertical position; tip submergence must be at the correct level beneath the liquid surface so as to avoid excess liquid coating outside the tip being inadvertently transferred during dispensing; and the samples must be mixed well.

Often, multiple replicates are needed for statistical inference and this requires the handling of multiple micro-centrifuge tubes and small wells in microplates. During the tedious qPCR sample preparation, mistakes are often caused by these challenging, concentration dependent and variable pipetting factors.

A robot that can handle all pipetting tasks like a human operator, without making any mistakes, whilst offering excellent reproducibility and accuracy, will eliminate these sources of error. Furthermore, it will significantly decrease the hands-on time required for scientists and technicians to prepare samples for qPCR experiments.

Andrew Alliance provides Andrew (Figure 1). Andrew is a pipetting robot and is the most easy-to-use and affordable automating solution for preparing qPCR reactions. Andrew can deploy the full volume range of its pipettes (from 0.2 µL to 1 ml) completely unattended, using conventional single channel pipettes designed for manual operations [2].

Andrew, the pipetting robot using conventional pipettes.

Figure 1: Andrew, the pipetting robot using conventional pipettes.

In combination with Andrew Lab, the graphical, user friendly software that enables users to easily design their own pipetting protocol, Andrew can smoothly execute all pipetting operations and incubations. These include: setting the volume, choosing the correct pipette for the volume, pre-wetting tips, inserting and ejecting tips, mixing by pipetting up and down, changing or keeping a tip etc.

This article describes the quantification of gene expression in human cells, using Andrew as an automating solution. The expression of three genes encoding the human necrosis factor receptor family members were specifically measured, and the Threshold Cycle (Ct) was calculated. This is the inverse proportion of the starting cDNA amounts in the sample. The pipetting accuracy in terms of linear regression from the Ct values of serial dilutions of the cDNA templates, and the pipetting reproducibility quantified by a coefficient of variation (CV) of Ct values from technical replicates, were also measured.

The same experiment was conducted manually and in parallel, using the same materials. This was to compare the performance of Andrew with the performance of a skilled human operator. Results show that Andrew reduces hands-on working time to more than half, as well as significantly improving data reproducibility and accuracy.

Andrew Reduces the Hands-On Time for qPCR Sample Preparation

Three members of the tumor necrosis factor receptor family were tested on a cDNA that was extracted from the spleen of a C57B/6J mouse:

  • BAFF Receptor (B-cell activating factor Receptor)
  • TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor)
  • BCMA (B cell maturation)

The positive control was ß-actin, and the negative control was water. Using the “Serial Dilution” wizard in Andrew Lab, the cDNA was diluted at 20, 100, 1,000 and 10,000 times. Next, 5 µl of each dilution was distributed in a 96-well PCR plate (Scientific Specialties Inc, Lodi, CA, USA). One tip was used for each dilution. Four different master mixes containing Taq Polymerase, IQTM SYBR® Green Supermix (Bio-Rad), forward and reverse primers of four different primer pairs for the four genes were prepared.

To each point of the cDNA dilution series, 13 µl were added. When added to the tubes or the wells, every reagent was mixed and the “high viscosity” option was selected to avoid bubbles during preparation and distribution of the master mix in the PCR plate. All reactions were tested in duplicate or triplicate to check the reproducibility of the pipetting operations.

It took 10 minutes to design the qPCR protocol in the Andrew Lab software. The protocol provides preview statistics about the numbers of pipettes and pipette tips required for organisation of and preparation for the experiments (Figure 2). It only took two minutes to manually place the consumables onto Andrew’s deck (Figure 3) and the protocol was executed by Andrew, unattended, without problems.

Statistics of pipettes and tips given in Andrew Lab.

Figure 2: Statistics of pipettes and tips given in Andrew Lab.

Top view of working deck of Andrew.

Figure 3: Top view of working deck of Andrew.

With manual operation, the hands-on work included five minutes of protocol design and 25 minutes of pipetting execution. Therefore, using Andrew reduces the total bench working time for users of this protocol from 25 minutes to two minutes (Figure 4).  

Andrew reduces hands-on time of qPCR sample preparation.

Figure 4: Andrew reduces hands-on time of qPCR sample preparation.

Andrew Prepares qPCR Samples with Excellent Accuracy and Precision

After the full experimental setup, done either manually or by Andrew, the plates were assayed in an iCycler iQTM Real-Time PCR detection system (Bio-Rad). Using serial dilutions of a reference cDNA preparation from murine spleen mRNA, standard curves were generated.

The Ct was calculated and analyzed for each data point. Ct is the intersection between an amplification curve and a threshold line and, as such, is a relative measurement of the concentration of the target in a PCR reaction. The lower the Ct value, the greater the amount of target nucleic acid present in the sample [3].

For the samples without cDNA template (negative controls), no signal was detected. This indicates that no cross contamination occurred during automated qPCR set-up by Andrew. For each gene, the linear regression of the qPCR data of the 4-dilution series was plotted. Trend lines were added in order to calculate the R2 (coefficient of determination). R2 is a statistical measurement of how well the regression line approximates the real data points.

The higher the value of R2, the better the accuracy. All qPCR data produced manually and by Andrew had R2 values of above 0.99. This indicates exceptional accuracy, and, in general, data from Andrew had higher values (Figure 5). These results show that the pipetting accuracy of the serial dilutions compared to those obtained manually is improved by Andrew.

Pipetting accuracy measured as linear regressions of Ct from qPCR of the dilution series of 4 genes produced by Andrew and manually.

Figure 5: Pipetting accuracy measured as linear regressions of Ct from qPCR of the dilution series of 4 genes produced by Andrew and manually.

The coefficient of variations (CVs) for the Ct duplicates and triplicates was calculated to evaluate the reproducibility of pipetting. The CVs of Cts obtained manually varied greatly between 0.07% and 0.3% whilst the CVs of Cts obtained with Andrew ranged from 0.11% - 0.17%. This suggests that Andrew’s pipetting is more consistent than the pipetting of the experienced human operator (Figure 6).

Pipetting reproducibility of Andrew and manually.

Figure 6: Pipetting reproducibility of Andrew and manually.

Summary

Andrew is the simplest to use and most affordable robotic platform for the setup of qPCR experiments. It maintains the supreme reproducibility and accuracy required for this type of sensitive quantitative assay. Using the pipetting performance of Andrew, scientists can easily standardize qPCR setup of their reactions without errors and cross contamination.

Additionally, the hands-on time required to prepare the experiment was significantly less (only 12 minutes for designing protocol and preparing consumables for Andrew to execute the pipetting). Using Andrew, laboratories can now significantly improve both lab efficiency and quality of downstream results without modifying existing workflows.

References

[1] Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Bio- technology. 1992, Vol. 10, 413-7.

[2] Andrew Alliance. Andrew, the pipetting robot using manual pipettes. www.AndrewAlliance.com [Online] 2012.

[3] Bustin SA1, Benes V, Garson JA, Hellemans J, Huggett J, Ku- bista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vande- sompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, Vol. 55, 611-22.

About Andrew Alliance S.A.

Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.

Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.

Start with meeting customer needs, end with customer feedback.

Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.


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Last updated: Jul 22, 2019 at 8:00 AM

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