Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy.
PLoS One. 2019 Sep 11;14(9):e0221923. doi: 10.1371/journal.pone.0221923. eCollection 2019.
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.
实用的实验室课程，教授在开发治疗策略中采用的分子药理学方法，对于生物技术、应用生物学、制药和技术化学、转化肿瘤学等课程的学生来说非常感兴趣。不幸的是，在大多数情况下，需要转移到学生学习的技术是复杂的，需要多步骤的方法。在这方面，简单明了的实验方案可能非常有趣。本研究旨在介绍一个实验室练习，重点（a）是一项极具挑战性的治疗策略，即 microRNA 治疗，（b）是应用生物学和药理学中非常感兴趣的生物分子，即肽核酸（PNA）。该实验的目的是确定：（a）PNA 介导的 RT-qPCR 可能出现的阻滞，最终用于证明 PNA 对选定 miRNA 的靶向作用；（b）突变 PNA 序列可能缺乏活性；（c）对其他不相关 miRNA 序列的扩增的影响（如果有）。可获得的结果支持以下结论：PNA 介导的 RT-qPCR 阻滞可以通过简单的方法进行分析；突变 PNA 序列完全没有活性；所使用的 PNA 的作用是特异性的，对其他不相关的 miRNA 序列没有抑制作用。该活性简单（细胞培养、RNA 提取、RT-qPCR 都是成熟的技术）、快速（从分离和表征的 RNA 开始，只需几个小时）、高度重现（因此即使是未经培训的学生也可以轻松使用）。另一方面，这些实验室课程需要一些设备，最关键的是需要有用于 PCR 的仪器。虽然如果这些仪器不可用，可能会成为一个问题，但我们想强调的是，使用基于琼脂糖凝胶电泳的标准分析方法也可以确定是否存在扩增产物。
