Quantitative polymerase chain reaction (qPCR or real-time PCR) is the gold standard for precise monitoring of selected genes. Its keen ability to both detect and simultaneously quantify specific DNA sequences underlies its popular use in diagnostics and basic research. Additionally, coupling qPCR with reverse transcription (RT-qPCR) provides a powerful means to quantify mRNA in cells or tissues.
The field of qPCR is experiencing a surge of interest and rapid expansion buoyed by advances such as instrumentation that pushes capacity to 1,536 wells and optimization-free multiplexing. Select Bioscience’s “Advances in qPCR” meeting, held recently in Berlin, showcased innovations in the field and provided expert advice for improving and enhancing qPCR.
Most biologic processes comprise a complex maze of interactions. Identifying the key players involved by utilizing real-time PCR can provide a signature of overall activity. “It is important to understand how the combined activity of many genes contributes to pathways related to health and disease,” said Kathy Lee, senior application specialist for genomic assays at Life Technologies.
“Genome-wide transcriptome studies are often employed to generate an initial list of genes that displays differential expression across treated and untreated tissues, or diseased versus normal tissues.”
New attention is being focused on identifying pathway-based biomarkers because they can be important diagnostic tools and predictors of treatment outcomes. “Finding biomarkers involves direct screening of genes from a specific pathway or a set of biologically or functionally related genes that hopefully provides a biological signature. We have developed more than 150 TaqMan® Gene Signature Arrays for human, mouse, and rat in both a 96-well format as well as a 384-well micro-fluidic cards.”
“It is critical that you trust your results,” Lee said. “The TaqMan Arrays allow reproducible and quantitative real-time, high-throughput screening for many samples and genes. Additionally, they allow a much higher sensitivity, specificity and dynamic range than just DNA microarrays.”
To achieve robust and meaningful results requires understanding the many sources of experimental variance in the qPCR workflow, reported Raza Ahmed, Ph.D., qPCR product specialist at Agilent Technologies. “With the drive to detect even smaller quantities of sample, it is crucial to address experimental variability at all stages. Many sources of error can appear such as in the experimental design, during sample preparation and purification, in the qPCR reaction itself, and during post-run analysis.
“Know your sample and its complexity,” Dr. Ahmed explained. “The complexity of a tissue affects the quantification results. For example, analyzing mixed cell populations could lead to underestimating the quantity of the sample. Nonpositive cells can dilute target concentration. Also, where a tissue is sampled is important. Thus, you must make sure your sampling does not introduce bias.”
Care should be taken from the very beginning of the process. “The quality of the template and any inhibitors present can lead to failure of detection,” Dr. Ahmed added. “Further, various compounds can be inhibitory such as phenols, cell debris, EDTA, lipids, and high amounts of RNA or genomic DNA. To get around these obstacles, samples need to be rerun at a higher dilution if possible. Including internal controls helps to monitor any sample inhibitors.”
Care should be exercised even during qPCR set up, according to Dr. Ahmed. “Optimize both your forward and reverse primer concentrations. Adjust the buffer conditions if needed. Take the time needed to optimize and validate your assays. The last thing you want is to have low efficiency and high variation. But with careful scrutiny you can produce reliable results.”