Demonstrating specificity of bioactive peptide nucleic acids (PNAs) targeting microRNAs for practical laboratory classes of applied biochemistry and pharmacology
Autoři:
Jessica Gasparello aff001; Chiara Papi aff001; Matteo Zurlo aff001; Roberto Corradini aff002; Roberto Gambari aff001; Alessia Finotti aff001
Působiště autorů:
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
aff001; Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy
aff002; Interuniversity Consortium for Biotechnology (CIB), Trieste, Italy
aff003
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0221923
Souhrn
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.
Klíčová slova:
Biology and life sciences – Biochemistry – Nucleic acids – RNA – Non-coding RNA – Natural antisense transcripts – MicroRNAs – Genetics – Gene expression – Gene regulation – Molecular biology – Molecular biology techniques – Artificial gene amplification and extension – Polymerase chain reaction – Research and analysis methods – Extraction techniques – RNA extraction – Research facilities – Research laboratories – Biological laboratories – Biological cultures – Cell cultures – Cultured tumor cells – Glioma cells – Database and informatics methods – Bioinformatics – Sequence analysis – RNA sequence analysis
Zdroje
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PLOS One
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