In cancer research, many biological aspects—from genes and molecules to cells and organisms—must be considered. Analyzing DNA is often part of the workflow. One of the most common techniques for studying DNA is the polymerase chain reaction (PCR)—especially the real-time polymerase chain reaction (qPCR), also known as quantitative PCR, hence the ‘q’. Making this technique more widely used and effective in cancer research depends on new techniques and tools.
Many scientists apply qPCR to cancer. In Italy, a team of scientists analyzed the expression of a cell adhesion molecule, L1CAM, in endometrial cancer. The results indicated that this molecule plays a role in the spreading of this cancer. As the scientists pointed out: “In pre-operative setting, L1CAM could be a useful additional tool. It could help to identify those … patients who are at high risk of disease progression.”
Many studies focus on breast cancer. The recurrence of any cancer can be very dangerous, because the disease that comes back is often resistant to treatment. With primary breast cancer, the expression of microRNAs (miRNAs) might indicate the likelihood of relapse. So, scientists in Greece tracked miRNAs in plasma with qPCR. The results indicated that levels of specific miRNAs could indicate the odds and timing of relapse, as well as the overall survival. The researchers even concluded: “Circulating miRNAs are differentially expressed among relapsed and nonrelapsed patients with early breast cancer and predict recurrence many years before its clinical detection.”
Other markers can also be used to assess the odds of breast-cancer relapse. One team of scientists used qPCR to see if induced macrophages impacted the growth of breast cancer cells. The results showed that even culture medium that had contained macrophages could inhibit the growth of breast cancer cells.
Image: Scientists use the real-time polymerase chain reaction (qPCR) to study many diseases, such as the breast cancer cells shown here, with DNA (blue) and a membrane protein (green) stained. Image courtesy of NCI Center for Cancer Research, National Cancer Institute, National Institutes of Health.
PCR can also be used to study the interaction of molecules related to cancer. For example, an international team of scientists used qPCR and other techniques to study what happens when a HOX protein—known to be expressed in various cancers—is blocked from binding to its cofactor. In describing the behavior of HOX in cancer, these scientists wrote: “The pattern of overexpression suggests that inhibition may be useful therapeutically.” In fact, this work showed that inhibiting that HOX-cofactor binding killed some cancer cells.
Rather than just looking at cells or molecules, even pieces of molecules can impact cancer, especially if those molecules are part of DNA. At the Indiana University School of Medicine in Indianapolis, scientists developed a qPCR-based assay for telomere length. As they wrote: “Excess telomere shortening has been observed in most cancer cells.” Despite the existence of a qPCR-based telomere assay, these scientists noted: “However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA.” This team’s new approach takes into account the multiple-copy sequences common in cancer. Consequently, the scientists conclude that their assay “would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.”
Many manufacturers are focused on advancing qPCR, often for cancer-related applications.
Droplet or not
When asked about the latest advance in qPCR that can be used to study gene expression related to cancer, Christian Nievera—senior product manager, molecular platforms at MilliporeSigma—says, “Digital PCR, especially droplet digital PCR, is the latest.” Nievera adds, “Also, if we consider next-generation sequencing (NGS) as a related method, then a key advance is RNA-sequencing for whole transcriptome gene expression profiling.”
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For droplet digital PCR (ddPCR), the benefits include “absolute quantification without a standard curve, better precision—that is, more reliably detecting small differences—than standard qPCR, better signal-to-noise ratio and easier quantification,” Nievera explains. No advance, though, tends to come for free. As Nievera adds about ddPCR, “You need special equipment and training to partition samples into thousands of tiny droplets, or create an emulsion.”
Nonetheless, Nievera notes that NGS is the “gold standard” for RNA sequencing. “A key advantage over ddPCR is the ability to multiplex samples and amplicons enabling simultaneous quantification of many templates from many sources,” Nievera says. “That said, for the standard qPCR workflow, MilliporeSigma has various qPCR kits along with the primers and probes as part of its comprehensive portfolio of PCR products.”
Picking a platform
However a scientist decides to take on cancer research, lots of options in qPCR can be considered. In fact, some vendors roll out complete families of PCR platforms. For example, Thermo Fisher Scientific makes the QuantStudio family, which includes a variety of qPCR platforms and even a digital one.
Consumables also impact the ability to use qPCR in cancer research. In London, scientists at BioLine developed the SensiFAST qPCR kits, which are “designed for superior sensitivity and specificity with probe-detection technology, including TaqMan, molecular beacon and Scorpions probes,” the company notes.
PCR primers can also be developed specifically for cancer research. As an example, System Biosciences used microRNAs that play a role in cancer to make qPCR primers that can be used with the company’s RNA-Quant cDNA Synthesis Kit and OncoMir qPCR Array. Together, says the company, this “provides a streamlined system for studying oncogenesis with qPCR.” In addition, System Biosciences notes that this technology can used to “simultaneously profile 95 different miRNAs known to be involved in apoptosis, differentiation, and cancer.”
Other commercial arrays for cancer can also be explored. The ExProfile Cancer Gene qPCR Arrays from GeneCopoeia, for instance, can be used to “profile the expression of cancer-related genes, which are carefully chosen for their close cancer correlation based on a thorough literature search of peer-reviewed publications,” as explained by the company. “Arrays are available for expression profiling of specific types of cancer-related genes.” This system comes in a 96-well format composed of as many as 84 PCR primers, plus a dozen control wells. According to GeneCopoeia, this product “detects as few as four copies of RNA using ExProfile qPCR array and recommended reagents/conditions.”
Pushing qPCR to track cancer more closely helps scientists explore more of cancer’s mechanisms and divide the disease more carefully based on molecular features. Ultimately, that should lead to better treatments that enhance and extend the lives of patients.