By Ben Butkus
Scientists from the European Molecular Biology Laboratory have devised a new method of profiling microRNA expression using quantitative real-time PCR, one of the technique's developers revealed at an industry event last week.
The method, called miQPCR, promises to improve upon current miRNA profiling techniques because it can universally profile all mature microRNAs in a sample in a single step; can quantify and discriminate between closely related miRNA family members; and is an open, adaptable, and cost-effective procedure, according to the researchers.
The group is now seeking collaborators wishing to help further develop or license and commercialize the method, which is patent-pending and has not yet been published in a peer-reviewed journal.
Mirco Castoldi, a postdoc with a joint appointment at EMBL and the department of pediatric oncology at the University of Heidelberg, unveiled some details about miQPCR for the first time in a presentation at Select Biosciences' combined qPCR/Epigenetics/RNAi/miRNA Europe conference in Munich last week.
According to Castoldi, even though several commercial miRNA-profiling methods exist, they are generally inflexible and have difficulties validating mature miRNAs. Current well-known commercial products include Life Technologies' TaqMan microRNA assays, Qiagen's miScript PCR system, and Exiqon's MiRcury LNA Universal RT microRNA PCR assays.
"A commercial platform needs to be universal; allow [melting temperature] adjustment of primers; and [be] open-ended," Castoldi said during his presentation. "Our system is an open method that can target any RNA in solution. One would have to use many different techniques to [profile] these different RNAs."
The miQPCR method works by taking advantage of the activity of single-stranded T4 RNA ligase to uniformly extend 3' ends of single-stranded RNAs with a specific RNA/DNA adapter, which the researchers have dubbed the miLinker.
"We have devised a specific sequence which should increase the process of elongation," Castoldi said. The researchers then use this linker to prime reverse transcription and cDNA synthesis during qPCR in order to detect the selected amplicon.
In an interview with PCR Insider following the conference, Castoldi noted that the addition of the miLinker in the miQPCR process allows reverse transcription of any RNA in a sample; thus, the group can theoretically use the method to detect any transcript.
The group has thus far developed dedicated protocols to design gene-specific primers targeting the three miRNA forms: precursor, primary, and mature miRNA; as well as piwi-interacting RNAs. Castoldi said that the group also has preliminary data demonstrating the ability to detect mRNA transcripts using miQPCR, but added that they are still optimizing primer design for mRNA target sequences.
One of the major advantages of the miQPCR approach is that it allows melting temperature, or Tm, adjustment of DNA primers. This is important, Castoldi explained, because the GC content across the "miRnome" is different than other transcript types, meaning that the predicted Tm for miRNAs varies between 40° and 80° C. In a typical qPCR protocol, the annealing and elongation steps occur at 60° C; thus, low-Tm transcripts may reduce assay sensitivity, while high-Tm transcripts may impact assay specificity.
"Exiqon resolves this problem by using [locked nucleic acid] nucleotides, [which are] expensive and the primer design is not intuitive," Castoldi said. "We do it by using the sequence of the linker. Indeed the linker elongates all the microRNAs in the samples and now the target sequences are longer than the … original microRNA."
By using this new sequence to design DNA primers, he added, the group can both adjust the primer Tm and ensure that the primer will be specific enough to discriminate between very closely related miRNAs — even those with only a one-base-pair difference in the middle of the sequence, according to initial studies conducted by the researchers.
The current miQPCR protocol takes about an hour and 20 minutes to complete; and an initial unpublished benchmark study shows that the method was at least as effective, if not more so, than Life Tech's TaqMan assays at analyzing expression of an identical panel of miRNAs in mouse heart and liver samples, Castoldi said. The group is currently withholding specific details of these studies as it prepares a manuscript for submittal to peer-reviewed scientific journals.
Castoldi unveiled the miQPCR method at the conference in hopes of drumming up interest from potential collaborators.
"We are looking for collaborators, perhaps industrial partners, on two levels," Castoldi said. "On one level, … maybe some company would be interested in partnering with us and basically granting us funding or people [to work] on it to develop different approaches, and [increase] the number of primers."
Alternatively, the group is interested in finding an industry partner that "might be interested in acquiring a license and introducing this technique in their own portfolio," he added. The method was developed in the Molecular Medicine Partnership Unit, a collaborative effort of the University of Heidelberg Children's Hospital and EMBL, the latter of which is handling collaboration and licensing requests.
In the meantime, Castoldi and colleagues are using miQPCR to investigate microRNA expression in the liver within the context of different diseases. The group recently discovered a liver-specific miRNA called mir-122 that is required to maintain constant levels of iron in mice, a discovery that was the subject of a paper recently published in the Journal of Clinical Investigation.
"At the moment we use [miQPCR] to continue to try to describe why this type of control would be required," Castoldi said.
In addition, he said that the group has "adapted [miQPCR] to measure levels of microRNA in the plasma and in the serum of patients. And we have a collaboration with a hospital in Ireland where we are going to [look at] patients with different diseases related to the liver and … basically screen whether we can stratify the patients' response to certain drugs based on microRNAs in their blood."
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