The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection
Abstract Objective 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 mim...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-11-01
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| Series: | BMC Research Notes |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13104-017-2930-0 |
| _version_ | 1818083242840948736 |
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| author | Patrick Winata Marissa Williams Eileen McGowan Najah Nassif Nico van Zandwijk Glen Reid |
| author_facet | Patrick Winata Marissa Williams Eileen McGowan Najah Nassif Nico van Zandwijk Glen Reid |
| author_sort | Patrick Winata |
| collection | DOAJ |
| description | Abstract Objective 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. Results 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. |
| first_indexed | 2024-12-10T19:34:53Z |
| format | Article |
| id | doaj.art-1ec61094c9e4447eb7456cedcae65da1 |
| institution | Directory Open Access Journal |
| issn | 1756-0500 |
| language | English |
| last_indexed | 2024-12-10T19:34:53Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj.art-1ec61094c9e4447eb7456cedcae65da12022-12-22T01:36:10ZengBMCBMC Research Notes1756-05002017-11-011011710.1186/s13104-017-2930-0The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detectionPatrick Winata0Marissa Williams1Eileen McGowan2Najah Nassif3Nico van Zandwijk4Glen Reid5Asbestos Diseases Research InstituteAsbestos Diseases Research InstituteUniversity of Technology SydneyUniversity of Technology SydneyAsbestos Diseases Research InstituteAsbestos Diseases Research InstituteAbstract Objective 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. Results 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.http://link.springer.com/article/10.1186/s13104-017-2930-0MicroRNAMimicRT-qPCRTargomiRMesomiR |
| spellingShingle | Patrick Winata Marissa Williams Eileen McGowan Najah Nassif Nico van Zandwijk Glen Reid The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection BMC Research Notes MicroRNA Mimic RT-qPCR TargomiR MesomiR |
| title | The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection |
| title_full | The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection |
| title_fullStr | The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection |
| title_full_unstemmed | The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection |
| title_short | The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection |
| title_sort | analysis of novel microrna mimic sequences in cancer cells reveals lack of specificity in stem loop rt qpcr based microrna detection |
| topic | MicroRNA Mimic RT-qPCR TargomiR MesomiR |
| url | http://link.springer.com/article/10.1186/s13104-017-2930-0 |
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