Aims: To identify differential expression of noncoding RNAs after trypsinization in human mesenchymal stromal cells (hMSCs), focusing on miRNAs, piRNAs and circRNAs. Methods: hMSCs from the bone marrow of three donors were collected for RNA extraction, either lysed directly in monolayer or trypsinized and lysed within 30 min. Total RNA was isolated and sequenced for the evaluation of miRNA and piRNA expression or RNaseR treated and labeled for circRNA array hybridization. RT-qPCR was performed to evaluate the stability of candidate reference genes. Results & conclusions: Alterations in levels of several noncoding RNAs are rapidly induced after trypsinization of hMSCs, affecting critical pathways. This should be carefully considered for a proper experimental design.
Keywords: RNA sequencing; cell detachment; circular RNA; human mesenchymal stem cells; loss of anchorage; microRNA; noncoding RNA; piwi-RNA; reference gene stability; trypsinization.