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University of Copenhagen Team Developing qPCR Assay to Measure Intracellular siRNA Delivery

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By Ben Butkus

A group of scientists from the University of Copenhagen has devised a qPCR-based assay to quantitatively measure the intracellular concentration of small-interfering RNAs and thus gauge the effectiveness of carrier-mediated siRNA delivery.

Although it is still in the testing phase, the researchers believe the tool will improve upon existing methods for assessing the ability of different types of nanocarriers to deliver siRNA payloads for RNA interference-based pharmaceutical applications.

Stefano Colombo, a PhD student in the faculty of pharmaceutical sciences at the University of Copenhagen, disclosed the method in a poster presentation last week at Select Biosciences' combined qPCR/Epigenetics/RNAi/miRNA Europe conference in Munich. Colombo's poster was judged by conference attendees as the best of those in the qPCR conference track.

As explained in the poster, therapeutic applications for RNAi depend on safe and effective carriers that can release chemically intact siRNA into the cytoplasm.

The University of Copenhagen research group has been studying and developing these delivery vehicles for the past few years, and was seeking a way to better evaluate which ones were doing their job, Colombo said.

"Screening the literature I found out that generally siRNA delivery efficiency is defined as the ability of carrier-mediated delivery to produce RNA interference," Colombo wrote in an e-mail to PCR Insider following the conference.

The RNAi effect, in turn, is typically measured by performing qPCR on target mRNA to assess transcript degradation; by measuring protein synthesis inhibition using flow cytometry on fluorescent protein assays; or by measuring cellular uptake with flow cytometry or confocal microscopy on labeled carriers, Colombo said.

However, "the role of … siRNA cellular delivery does not end with the uptake," Colombo said, noting that it is only one step of siRNA delivery, which depends on not only carrier action but also cellular processes.

Starting from this idea, the group decided to pursue a new approach that would define drug delivery efficiency as the ability of a carrier to produce a concentration of siRNA in the cell.

In order to unambiguously compare the efficiency of different carriers, the measurement technique "must be quantitative and must be as much as possible independent from the cellular activities not related to the carrier-mediated delivery process," such as mRNA degradation and translation, Colombo said, adding that "the most convenient and sensitive technique identified to measure intracellular intact siRNA is qPCR."

Colombo and colleagues adapted a universal stem-loop qPCR assay previously described by two different groups. The first assay was developed by a group of researchers from Life Technologies led by Caifu Chen, who has been involved with the development of several novel PCR techniques at Life Tech and predecessor Applied Biosystems.

Chen and colleagues described this assay and its application for quantifying microRNAs and siRNAs in a 2005 Nucleic Acids Research paper, and it has become the basis of a commercial assay from Life Technologies.

The Copenhagen researchers also referenced a universal stem-loop assay described by researchers from the RNA Therapeutics department at Merck in a 2008 Analytical Biochemistry paper.

According to Colombo, the major drawbacks of the stem-loop assay as originally described were low specificity and the unwanted formation of dimers and other structures that impair assay efficiency.

The Copenhagen group addressed these drawbacks with several modifications to adapt the stem-loop assay for their research. For instance, they designed a combined DNA/locked nucleic acid hydrolysis probe "to be specific and bright," Colombo said. "It is short — 12 [base pairs] — but has a high Tm. Using special patterns of DNA/LNA we introduced some sites known to be very mismatch-sensitive. This feature made the probe less prone to produce unwanted interactions that can reduce the assay efficiency."

In addition, the researchers modified the stem-loop primer backbone sequence to reduce self interactions; and made some modifications to preserve the thermodynamic stability of the stem-loop structure. "The final result is a decreased formation of dimers and other structures that could impair the reaction efficiency," Colombo said.

So far, the group has tested its assay on commercial transfection systems as proof of principle and achieved positive initial results, but has not yet identified a potential siRNA carrier for study.

"The technique is still in a testing phase," Colombo said. "Our aim is to improve our understanding of siRNA delivery by this method to be able to improve the mechanistic knowledge of our carriers." Specifically, the group is developing nanocarriers such as PLGA nanoparticles and dendrimers for therapeutic siRNA delivery.

The researchers have not thought about patenting the method yet, Colombo said, adding that they believe the approach "can be related to commercial technologies and maybe will be useful in assessing commercial transfection reagents or treatments."

He noted that he and his colleagues are conducting additional research "to obtain results that could be useful for [companies] that work in the drug-delivery field or siRNA technology."


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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