FISH, qPCR, microarrays, and NGS are the main technologies for studying genomic abnormalities in clinical applications. Here, let’s look at FISH and qPCR. Where do they stand out? What are their weaknesses? What does the future hold for them?
What is qPCR?
Quantitative polymerase chain reaction (qPCR) is a variation on the original PCR method developed in the 1980s that uses fluorescent probes to detect amplicons as they amplify. In diagnostics, qPCR assays can help determine the presence of genetic markers in real time, and therefore aid disease diagnosis.
qPCR is inherently analog (i.e. the output is continuous), and quantitation relies on setting a threshold against standards. Digital PCR, (dPCR) is a variation on qPCR that dilutes a single sample into many smaller reactions. Each reaction contains no more than one template molecule. Therefore, amplification can give you a discrete and absolute count of how many template molecules were present in the original sample and, importantly, a definitive answer on the presence of a marker.
qPCR or RT-qPCR?
If you’re interested in profiling gene expression rather than the gene itself, a variation of qPCR—reverse transcriptase qPCR (RT-qPCR)—enables you to analyze the relative abundance of RNA. The reverse transcriptase converts the RNA to cDNA, which you can then treat in essentially the same way as a DNA sample.
Pros and cons of qPCR and RT-qPCR
qPCR has been around since the 1990s and has remained the most popular method in molecular diagnostics, despite the development of more modern approaches. Its continued success is largely the result of its four key benefits: sensitivity, simplicity, speed, and cost.
Compared to other diagnostic assays, qPCR is a relatively cheap way to study genomic ‘targets’, even at tiny concentrations, with several reagents common to standard PCR, such as the polymerase and nucleotides. A typical protocol is also fast—often providing same-day results—and the data is easy to interpret.
A key drawback when choosing qPCR is that it isn’t really designed for interrogating complex or multi-gene conditions. You can only use so many probes in one assay, and so investigating many targets substantially increases PCR workload. This then affects costs and turnaround times.
Another downside to PCR is the potential for biased outcomes. The approach requires pre-selection of targets, so the outcome of any assay is limited to the probe selection for that assay. Co-infections, for example, are easy to overlook with primers designed to pick up one infection and not another.
Clinical applications for RT-qPCR
Clinicians frequently turn to qPCR or RT-qPCR to identify pathogens; together, they make up more than 50% of the diagnostic testing area. Infectious disease diagnosis (i.e. the identification of bacteria or viruses) can involve both DNA and RNA analyses. This often requires fast results, making RT-qPCR well suited.
In human genetics, key areas for PCR are oncology and reproductive health. Clinicians can use PCR to detect, for example, minimal residual disease in leukemia patients, either through DNA or RNA. In reproductive health, PCR can pick up chromosomal aberrations at the preimplantation (in in vitro fertilization), prenatal, or postnatal stage of development.
The future of clinical qPCR
The overall trend in clinical DNA analysis is one of increasing scale. This trend is driven mainly by ever-decreasing costs of DNA sequencing. As a result, NGS-based assays are likely to replace PCR in applications where information from large or multiple regions of the genome determines diagnosis.
Still, qPCR and RT-qPCR retain a large market share in molecular diagnostics and are likely to remain key players for the foreseeable future, particularly in applications where rapid results are critical. This need is most clear in the infectious disease market.
The simplicity of qPCR also makes it suitable for miniaturization, which, in combination with speed, could make qPCR a viable alternative to sequencing.
What is fluorescence in-situ hybridization (FISH)?
FISH is a cytogenetic technique for visualizing sequences of DNA or RNA in-situ within the cell and tissue environment (e.g. a tissue section on a microscopy slide). It works by adding to your sample fluorescent probes that only bind to a specific nucleotide sequence. The fluorescent signal will tell you whether your sequence is present—and how much of it is there.
Pros and cons of FISH
Like PCR, FISH is a relatively easy-to-use and inexpensive technique. Of the four techniques I look at in this series, FISH is the also only one that gives you images rather than digital readouts.
For this reason, clinicians can use FISH to look at where relevant sequences are located, which is helpful for studying genomic translocations and heterogeneous tissue samples, such as tumors.
FISH analysis is also well suited for formalin-fixed paraffin-embedded (FFPE) samples, commonly used in many clinical settings. Other methods for analyzing genetic material sometimes struggle with FFPE because of the DNA damage introduced during processing, but the high sensitivity of FISH assays means it can give clear results in FFPE.
However, as with PCR, the options for studying multiple areas of the genome simultaneously are limited. It’s possible to treat slides with multiple probes, but only as long as you can reliably distinguish the different colors under the microscope.
Another drawback of FISH is that it’s limited to large genetic aberrations, such as copy number variation (CNV). For looking at smaller aberrations, such as single-nucleotide polymorphisms (SNPs), you’ll likely need to use a different technique.
FISH in the clinic
Key areas for FISH-based analysis include oncology and constitutional genetic testing. In cancer diagnostics, FISH is popular for studying large cancer-causing mutations (e.g. CNV) and chromosomal rearrangements (e.g. the BCR/ABL translocation in leukemia).
In reproductive health, FISH is well suited for the detection of extra chromosomes in babies or fetuses with trisomy, such as Down syndrome (trisomy 21), as well as a range of other developmental disabilities, such as DiGeorge syndrome (22q11.2 deletion syndrome).
The future of FISH-based analysis
Moving forward, the growth of FISH-based analysis is likely to slow down as it loses ground against DNA sequencing due to decreases in NGS costs and improvements in detection of, for example, CNV. NGS is becoming more established in cancer diagnosis and monitoring, as well as in reproductive health applications.
Nevertheless, the ability to visualize a sequence within the context of its surroundings remains a substantial benefit for studying heterogeneous samples. It means that FISH still has the potential for continued success in the future, even as larger-scale genomic analysis is becoming the norm.
If you missed part one of this series, check out two other common molecular diagnostic techniques—microarrays and DNA sequencing—designed for looking at large areas of the genome.
To find out more about challenges and solutions in genomics, take a look at our other blogs. For support with optimizing your genomic analyses, contact Cytiva Support or your local Cytiva representative.