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Demonstrating specificity of bioactive peptide nucleic acids (PNAs) targeting microRNAs for practical laboratory classes of applied biochemistry and pharmacology.
- Source :
-
PloS one [PLoS One] 2019 Sep 11; Vol. 14 (9), pp. e0221923. Date of Electronic Publication: 2019 Sep 11 (Print Publication: 2019). - Publication Year :
- 2019
-
Abstract
- Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis.<br />Competing Interests: The authors have declared that no competing interests exist.
- Subjects :
- Cell Line
Cell Proliferation drug effects
Gene Expression Regulation drug effects
Humans
Mutation
Peptide Nucleic Acids genetics
Reverse Transcriptase Polymerase Chain Reaction
Biochemistry education
MicroRNAs antagonists & inhibitors
Peptide Nucleic Acids pharmacology
Pharmacology education
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 14
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 31509554
- Full Text :
- https://doi.org/10.1371/journal.pone.0221923