Polymerase chain reaction (PCR) was developed in 1983, and since then, it has become an essential tool for various applications in medical and life science research laboratories. Together with fluorophores, the PCR also serves as a direct, quantitative reporting mechanism for the abundance of a target transcript in a sample (quantitative real-time PCR, qPCR).
This insight can be used to diagnose cancers, infectious diseases, and genetic abnormalities, and also to investigate food samples for contamination with genetically-modified organisms or bacteria. Since the technique has been widely available for more than three decades, a surplus of different reagent sets is available.
However, these reagent sets have one thing in common – they constitute a major cost of PCR experiments. Therefore, assay miniaturization offers a huge scope for cost savings, but miniaturization means minimum volume that can be reliably used to achieve reproducible results.
This article shows how the Tecan D300e is used to set up qPCR reactions using different master mixes and reaction volumes, and then compared to manual reaction set-up.
Scaling down an assay means a trade-off between reagent savings and reliability of the results. Therefore, the precision and efficiency of the qPCR reactions was compared at different reaction volumes.
A 384-well plate was used to pre-dispense the primers, and this was followed by dispensing the Precision Melt Supermix (Bio-Rad) at different volumes (1, 1.5 and 2.5 μl). Next, 1 μl of cDNA (1 ng/μl) consisting of 0.1% TritonTM X-100 was dispensed into all reaction wells.
An additional 5 μl of 1:1 cDNA (0.4 ng/μl + 0.1% TritonTM X-100) and master mix was also dispensed into all of the wells to obtain a total of 1 ng of DNA per reaction. Three replicates were carried out for each point, and the results show that similar cycle thresholds (Cts) are obtained for reaction volumes as low as 2 μl (Figure 1).
Figure 1. Comparison of Cts for different reaction volumes showing similar results for volumes down to 2 μl. Triplicates were performed for each sample and error bars represent standard deviations.
Reactions that were set up with the Tecan D300e were also compared with those prepared manually. Fast SYBR® Green (Thermo Fisher Scientific), master mix was added into the 384-well plate, which had been pre-plated with primers.
Subsequently, samples were manually added. Tween® 20 or TritonTM X-100 was added to obtain final concentrations of 0.3% and 0.1%, respectively, in total reaction volumes of 5 μl, and for each point, three replicates were done.
The results indicate a good correlation between the automated set-up and the manually pipetted set-up, and also demonstrate that a detergent is not required for reliable dispensation of the master mix (Figure 2).
Figure 2. Comparison of Cts for reaction set-up by hand or by D300e with or without the addition of detergents in total reaction volumes of 5 μl. Fast SYBR® Green (Thermo Fisher Scientific) master mix was used and triplicates were performed for each sample and error bars represent standard deviations.
In order to compare different master mixes, the experimental set-up was conducted in 5 μl reaction volumes (as described earlier) using Precision Melt Supermix (BioRad) and Brilliant III Ultra-Fast SYBR® (Agilent Technologies).
The results obtained by automated set-up and manual set-up were found to be similar, without any major difference (Figure 3), although specific transcripts seem to be more easily detected by one master mix than the other.
Figure 3. Comparison of Cts for reaction set-up by hand or by D300e in total reaction volumes of 5 μl. Brilliant III Ultra-Fast SYBR® (Agilent Technologies) and Precision Melt Supermix (BioRad) were used, triplicates were performed for each sample and error bars represent standard deviations.
Reagents from four different suppliers were used to further investigate the performance of the different master mixes. As before, the primers were pre-plated except this time the samples were dispensed with surfactant (0.1 % TritonTM X-100), and the master mixes were dispensed without adding a surfactant.
This time too, triplicates were carried out for each point. From the results, it becomes clear that the master mixes demonstrate mostly comparable performance in detecting the test transcripts (Figure 4).
However, these experiments did not help determine whether the high Ct values for the SYBR select master mix is the result of this reagent being more sensitive to detergents, or due to lower general performance. Despite this fact, the sensitivity of the reagent of choice to detergents should be taken into account for all reaction set-ups.
Figure 4. Comparison of Cts for reaction set-up using the D300e in total reaction volumes of 5 μl. The master mixes are indicated in the figure. Triplicates were performed for each sample and error bars represent standard deviations.
The data presented in this article shows that the Tecan D300e can be effectively employed to miniaturize qPCR reactions to volumes down to 2 μl, which is not possible with a manual pipetting set-up. Other than the reagent savings obtained through miniaturization, the greater speed and process security of the automated set-up is more beneficial for genomics workflows.
Data courtesy of Robbie Allen and Dylan Nelson from Oregon Translational Research and Development Institute, Portland, USA.
Produced from materials originally authored by Dr. Manuel Bauer, product manager, Tecan.
Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.
In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.
They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.
They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.
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