Title |
The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection
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Published in |
BMC Research Notes, November 2017
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DOI | 10.1186/s13104-017-2930-0 |
Pubmed ID | |
Authors |
Patrick Winata, Marissa Williams, Eileen McGowan, Najah Nassif, Nico van Zandwijk, Glen Reid |
Abstract |
MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 18 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 4 | 22% |
Student > Bachelor | 2 | 11% |
Professor | 2 | 11% |
Other | 1 | 6% |
Student > Ph. D. Student | 1 | 6% |
Other | 3 | 17% |
Unknown | 5 | 28% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 8 | 44% |
Agricultural and Biological Sciences | 3 | 17% |
Medicine and Dentistry | 2 | 11% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 6% |
Unknown | 4 | 22% |