Large numbers of drug discovery projects involve the analysis of gene-expression levels to accurately assess target effects. When screening compounds from a library, it is common to use microarray techniques since they provide a broad overview of genome-wide expression levels. Microarrays do have their limitations, however—while they can generate a broad portfolio of data on a single chip, there are sometimes discrepancies over the precision of the expression levels of each individual gene.
Before progressing along the drug discovery pipeline, it is key that microarray results are effectively validated. Real-time quantitative polymerase chain reaction (qPCR) is often used as the final step in any microarray protocol to ensure the accuracy and repeatability of the data prior to further analysis.
The drug discovery process in its entirety can be both time-consuming and costly. In order to streamline this into an efficient workflow, accuracy at every step is essential. The most promising initial hits need to be identified and the least promising candidates eliminated early on. Therefore, microarray data needs to be reliable, and validation via qPCR is a logical quality-control step.
However, qPCR itself has its own challenges, with well-to-well and plate-to-plate variability impacting the accuracy of the quantification of expression. Users need to be confident that reaction uniformity is maintained across each plate throughout an entire PCR run.
In this article, we discuss the use of two Thermo Fisher Scientific products—the Thermo Scientific RNA Spike Control and PikoReal Real Time PCR thermal cycler—as part of a molecular biology workflow to reduce variability when amplifying target sequences.
qPCR relies on the versatility and resilience of the polymerase and primers to produce reliable and consistent results. Invalid qPCR data may be the result of a variation introduced from one of the many steps in the workflow, and a single anomaly can invalidate an entire compound screen.
Users consequently need to ensure that the samples are not contaminated with inhibitors such as salts (NaCl and KCl), detergents (sodium deoxycholate, sarkosyl and sodium dodecylsulfate), and organic solvents (ethanol, isopropanol and phenol), which will essentially decrease their effectiveness.
All of these compounds are used in the purification and isolation of nucleic acids from tissues and cells and can cause a significant reduction in the efficiency of the PCR reaction if not adequately removed.
Ahead of amplification, two RNA samples were analyzed: sample A was uncontaminated; sample B was contaminated with probable inhibitors. The two samples were then evaluated using the Thermo Scientific RNA Spike Control. The spike control molecule was added to either an RNA sample or a pure water sample.
Two routine reverse-transcription qPCR protocols were subsequently run in parallel, one using an aliquot from the RNA/spike molecule sample and one from the pure water/spike molecule sample. The results from both assays are shown in Figure 1, where the difference in Cq values suggests that an inhibitor is present within sample B.
In order to obtain both reliable and repeatable data, uniformity across the PCR plate is essential. By reducing variation between on-plate replicates, melting curve data is improved and quantification is more accurate, thus making the data for analysis more consistent. The PikoReal thermal cycler was tested to assess its ability to provide uniform heating across an entire 96-well plate (Figure 2).
Obtaining such clear uniformity is most essential for highly sensitive assays, where a specific annealing temperature is required and reactions can fail if the temperature deviates from the ideal, significantly impacting the efficiency and specificity of the reaction and potentially bringing the accuracy of the quantified data into question.
Ninety-six replicate reactions were set up in a 96-well plate, with 125 pg/5 µL of Lambda DNA and Thermo Scientific DyNAmo Color Flash SYBR Green Master Mix in each well. A recommended qPCR protocol was performed using the Thermo Scientific PikoReal thermal cycler. Temperature and Cq uniformity were subsequently analyzed across the plate. Cq values were subtracted from the average value of 96 replicates and plotted by well position, as shown in Figure 3.
By providing consistent data across the plate, users can be sure that their qPCR results are a reliable indication of which compounds can be moved forward in the drug discovery pipeline for further analysis.
Multiple Targets in One Well
Multiplexing can be used to provide high-throughput capabilities, as the expression profiles of multiple genes can be examined within one well using different fluorophores. This provides a wealth of expression information within a single qPCR run, saving significant laboratory time. Thermal cyclers such as the PikoReal contain five channels, which enable the fluorescence detection of multiple dyes within the same well. During a four-color hydrolysis probe assay, the PikoReal was used to successfully generate uniform, reliable, and repeatable data (data not shown).
The initial screen of a compound library is essential to determining the subsequent success of the drug discovery initiative. Commonly employed microarray techniques, however, can prove problematic, with the reliability and accuracy of the expression data of individual genes being brought into question. By incorporating a validation step, through the use of qPCR, this potential issue can be eliminated.
In order for this validation to be highly accurate, the margin of error for the qPCR data must be minimal. Inter-plate uniformity is critical to obtaining a reliable qPCR performance. Therefore, the thermal cycler must be able to maintain accurate temperatures across the plate throughout the different phases of each PCR cycle.
The sensitive nature of qPCR protocols means that small variations in Cq can generate large inaccuracies, creating the potential for ideal drug candidates to be overlooked for further development. Through the use of the Thermo Scientific RNA Spike Control and PikoReal Real Time thermal cycler, molecular biology workflows can be executed in a highly efficient manner.
Uniformity across the plate is crucial for primer probe annealing and melt-curve analysis. By having a highly uniform block at all temperatures, the PikoReal ensures accuracy of data for all applications and chemistries that may be required, regardless of plate position.
Furthermore, this thermal cycler reduces energy, plastic, and reagent consumption, supporting efforts of the growing number of environmentally conscious laboratories. By combining an environmentally friendly design with thermal uniformity and a small laboratory footprint, researchers can take advantage of “green PCR” while minimizing variations in their workflow.