The advantages and disadvantages of real-time PCR and NGS
As new technologies and methodologies emerge to address applications such as gene expression, it can get quite confusing to determine which approach is the best, whether one simply replaces the other, or do they work together to tell a more complete story? Here we try to demystify how to choose between real-time PCR (qPCR) and next-generation sequencing (NGS), or whether you really need to choose at all.
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Real-time PCR and whole transcriptome NGS
There are two primary approaches for whole transcriptome NGS: RNA-Seq and targeted transcriptome. RNA-Seq is recommended when one or more of the following are key to your research efforts:
- You want to detect all cellular RNA: mRNA, miRNA, tRNA, etc.
- You want to look at transcript isoform diversity/expression
- Novel discovery is critical, i.e. you want to identify differentially expressed genes without a priori knowledge of which targets might be impacted by your future treatment
Targeted transcriptome approaches are similar in that novel discovery is possible, although the output will be limited to the most common mRNA transcripts, generally in the range of 20,000 targets spanning most of the known transcriptome.
Where does real-time PCR fit into these workflows? Data integrity is key to gene expression experiments, therefore it is common practice to use real-time PCR both upstream and downstream of NGS. This means that we don’t live in a “real-time PCR vs. NGS” world; rather the two technologies are complementary, working together to generate trustworthy results. Upstream of NGS, it is common to use TaqMan real-time PCR to check cDNA integrity prior to NGS.
Downstream of NGS, real-time PCR continues to be the go-to method for verification of results. In cases when verification is unnecessary, it is important to note that real-time PCR is the gold-standard technology for follow-up studies involving a targeted panel of transcripts discovered during the NGS screen.
The publications below feature Ion AmpliSeq NGS accompanied by TaqMan real-time PCR for either cDNA integrity checks or verification of results.
BIOZ biomedical research articles using both Ion AmpliSeq and TaqMan technologies
Real-time PCR and Illumina targeted amplicon RNA-Seq
Illumina targeted amplicon RNA-Seq is an NGS method for measuring 12-1,200 transcripts of interest. This range of targets overlaps with what is possible with real-time PCR (Figure 1). The choice between real-time PCR and Illumina targeted amplicon RNA-Seq should take into account these factors:
- Historically, real-time PCR has been considered the gold standard for gene expression quantification. For this reason, real-time PCR instruments are ubiquitous, and the benchtop and data analysis workflows are familiar and straightforward. For an experiment involving 20 samples and 10 targets, with RNA samples and qPCR assays already in the freezer, all data can be generated in a few 96-well plates run across 1–2 days. The same experiment using Illumina targeted amplicon RNA-Seq would take longer, especially if an Illumina instrument was not easily accessible. Sending samples to a core facility or service provider would likely increase the sample-to-answer turnaround time.
- Illumina targeted amplicon RNA-Seq will also cost more money. Targeted amplicon NGS is more expensive for experiments involving less than 20 targets.
- Lastly, sensitivity and dynamic range are often cited as strengths of Illumina targeted amplicon RNA-Seq over real-time PCR given the absolute read-counting nature of NGS. It is worthy to note that in most experimental contexts, the sensitivity and dynamic range of real-time PCR are sufficient, and achieving top-level sensitivity in Illumina targeted amplicon RNA-Seq often requires sequencing to great depths, which drives up the cost of the experiment.
Frequently asked questions
Yes, TaqMan Gene Expression assays are available for most exon-exon junctions for all genes and species in our predesigned assay collection. To select an assay that is variant-specific, first identify the NCBI RefSeq transcript accession number, and then use that accession number as your search term in the TaqMan Assay Search Tool. In the resulting list of assays, choose the assay that detects only that one variant of interest. If all available assays detect more than one RefSeq transcript, then use our Custom Assay Design Tool to attempt a variant-specific assay design.
Yes, TaqMan Array plates are 96- and 384-well plates containing 8–384 assays (pre-spotted and dried down, all you have to do is add cDNA and master mix). Based on the number of samples and targets needed, order just enough plates to build in technical and biological replication. TaqMan Array cards are 384-well microfluidic cards containing 12–384 assays with the added benefit of using less master mix than conventional plates and a super-easy benchtop workflow: pipet 384-wells in 2 minutes. TaqMan OpenArray plates are the highest-throughput option for qPCR offering the lowest price per data point.
It actually depends. The number of samples and targets possible in a single Illumina targeted amplicon RNA-Seq run depends on the Illumina platform and chemistry. Assuming the desired number of samples and targets can fit in a single Illumina targeted amplicon RNA-Seq run, then it will take two days from sample prep to data analysis. If you have to outsource your Illumina NGS experiment to a core facility or service provider, then it can take days or weeks to get your results. In contrast, the following real-time PCR experiments are possible in 1–3 days:
For Research Use Only. Not for use in diagnostic procedures.