When it comes to real-time PCR, there are two schools of thought. Some investigators insist that probe-based assays are essential to obtain adequate specificity. Others feel SYBR green is more than adequate and gene-specific probes are a waste of time and money. The Universal Probe Library (UPL) offers a compromise that provides the specificity of a probe but does not require a new probe for each transcript. Roche’s claim that the library offers probe specificity at SYBR green prices is a bit exaggerated, but many investigators could save time and money with this system.
The principle behind the probes is that they include a locked nucleic acid that increases binding specificity and melting temperature, making it possible to use shorter probes of 8 or 9 nucleotides. The probe sequences are common in the transcriptome, allowing each probe to work for many transcripts. When a UPL probe is used with gene-specific primers, the probe only binds to the amplicon of interest, offering the specificity of a standard hydrolysis probe. The libraries have been selected so that 90 probes will cover the entire transcriptome of an organism.
My initial thought was that the probes were just as expensive as TaqMan® probes so it did not seem like a wise investment. However, I realized that some of our studies are very small and it would be great if we had probes which could be used with multiple primer sets. Our lab purchased about ten individual probes from the library. Using the assay design program on the Roche website, we tried to design new assays using the set of probes that we already owned. However, our lab does a lot of quantitative PCR so we ultimately decided to purchase the whole human library. The library seemed like a large investment, but it is really not that much compared with what we have spent on probes (and shipping) in the past. Now we just have to order a standard set of primers and we are ready to begin. Although off-the-shelf assays from other companies are just as fast, I often needed custom probes that could take a few weeks to synthesize.
The assays work with minimal optimization, unlike probes produced by an oligonucleotide vendor. A few years ago, I ordered dual-labeled probes and found that I had to titrate each probe and each primer pair to find the optimal combination. Pre-designed assays have nearly eliminated this optimization and I was initially concerned that I would be returning to a situation where I would again have to spend a lot of time and money optimizing, thus negating any savings. Fortunately, assays with Roche’s UPL probes typically work with a standard protocol and rarely require extensive optimization.
One slight disadvantage is that the UPL does not offer the convenience of having the primers and probe mixed together. However, by ordering the primers yourself, there are no issues with proprietary sequences. This issue surfaced when I wanted to clone TaqMan® amplicons to create standards and realized that I had to use the expensive probes as primers because I did not know the primer sequences. Another issue to consider is that each species has a separate library, although it is possible that complete probe sets will be available in the future. In our lab, we use human, rat, and mouse cDNA for our assays, but we only have a complete human library. We can still design rodent assays using the human probe library, but with some limitations.
Ultimately, the degree to which the library will be useful and cost-effective depends on the number of genes and samples that a lab processes. Labs that examine only a few genes in large numbers of samples can use UPL probes but probably won’t save much money. On the other hand, labs that examine a large number of genes in a smaller number of samples will avoid buying expensive probes and only using a small portion of each one.
Tara Lauriat
Graduate Student
Mount Sinai School of Medicine