Researchers from SomaGenics plan to use a $300,000 grant from the National Cancer Institute to develop a method they call mRNA Fragment Quantification, or mR-FQ, which they believe can overcome issues that standard RT-qPCR methods have with fragmented mRNA extracted from formalin-fixed paraffin-embedded tissue samples, hopefully allowing higher sensitivity and specificity.
In an abstract describing the project, the company outlined its plans to demonstrate a proof of concept for the method and show its superior performance in comparison to conventional RT-qPCR gene expression assays.
The company wrote that following its initial demonstration of the method, it hopes to then use a second phase of the project to create mR-FQ assays for "all established mRNA biomarkers for breast cancer," and integrate these into a multiplex PCR array for simultaneous expression profiling.
According to SomaGenics' vice president for discovery research, Sergei Kazakov, the team believes that the increased sensitivity of mR-FQ may allow researchers or diagnostics companies to increase the accuracy of current RT-qPCR FFPE gene expression signatures, and potentially to add additional mRNA biomarkers that can't currently be reliably detected in standard RT-qPCR tests.
Kazakov told PCR Insider this week that the approach could also be useful for gene expression profiling in other types of limited or degraded samples in areas like forensics or anthropology. As the company works to demonstrate the method, it plans to initially focus on breast cancer markers like HER2, but Kazakov said mR-FQ could be applied to biomarker discovery or testing in other cancers as well.
"HER2 mRNA is just a model used in our … project to demonstrate proof of concept," he explained via email.
However, he said, SomaGenics believes that mR-FQ assays could eventually augment or potentially replace the conventional RT-qPCR that is currently used in gene expression-based tests like Genomic Health's Oncotype DX.
In its grant abstract, SomaGenics wrote that the company hopes to eventually co-develop mR-FQ-based breast cancer companion diagnostic assays through collaborations with "leading pharma and diagnostic companies."
Kazakov explained that the choice of RNA biomarkers for many companies is currently limited by what RT-qPCR can and cannot reliably measure.
"Sometimes there are good mRNA biomarkers, but they are low-abundance, and they can't reliably detect expression by their assay, so they exclude them from the test," he said. "So our initial approach would be to assay the same mRNA biomarker sets that [these companies] already have and then try to really expand it because we would have potentially much higher sensitivity."
Potentially, "this could either extend the detectable mRNAs or maybe even dramatically change the biomarker set [a test] uses in some cases," he said.
According to Kazakov, mR-FQ is designed to overcome the struggles RT-qPCR has with the random fragmentation that occurs in RNA extracted from FFPE samples.
He declined to describe the mR-FQ approach in detail, but said that unlike standard PCR methods, the approach "explicitly makes use of mRNA fragmentation rather than avoiding it."
According to Kazakov, RNA fragmentation has been previously employed in RNA sequencing and microarray methods, but has not been used for RT-qPCR.
"Because of this random fragmentation, only a part of the mRNA sequences can be amplified [with current methods] because a single RNA cleavage anywhere between two preset PCR primers prevents PCR amplification, thus compromising the sensitivity of the assay," he said.
"The yield and length of RNA fragments that can be extracted from FFPE blocks can vary substantially from sample to sample, further limiting the consistency of data derived using current RT-qPCR assays," he added.
In its abstract describing the project, the company wrote that mR-FQ involves purifying RNA fragments of a certain size range, modifying these fragments to allow more efficient reverse transcription and simultaneous pre-amplification of target mRNA sequences, and a "novel PCR primer design" using single-dye readout.
Kazakov said the firm could not provide further details on how the RNA fragments are modified or how its PCR primer design allows increased sensitivity and specificity using a single dye.
He said the company has some preliminary results that it used to pursue the SBIR funding, but has not yet demonstrated a proof of concept for mR-FQ.
The initial phase one Small Business Innovation Research grant will last one year, Kazakov said. If the company's proof of concept is successful, he said SomaGenics would expect to move toward commercializing the technique within the next few years.