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Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells
Published on: March 8, 2019
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Cell detachment rapidly induces changes in noncoding RNA expression in human mesenchymal stromal cells
Elena Della Bella1, Martin J Stoddart1
1AO Research Institute, AO Foundation, Davos, Switzerland.
Biotechniques
|October 18, 2019
Summary
Trypsinization rapidly alters noncoding RNA levels in human mesenchymal stromal cells (hMSCs), impacting key cellular pathways. Researchers must consider these changes for accurate experimental design and results.
Area of Science:
- Molecular Biology
- Cell Biology
- Genomics
Background:
- Noncoding RNAs play crucial roles in cellular regulation.
- Mesenchymal stromal cells (hMSCs) are widely used in regenerative medicine and research.
- Cellular manipulation techniques, such as trypsinization, can influence gene expression.
Purpose of the Study:
- To investigate the differential expression of noncoding RNAs (miRNAs, piRNAs, circRNAs) in hMSCs immediately following trypsinization.
- To understand the impact of trypsinization on noncoding RNA profiles in hMSCs.
Main Methods:
- Human bone marrow-derived hMSCs from three donors were used.
- Cells were either lysed directly or after trypsinization within 30 minutes.
- RNA was extracted and subjected to sequencing for miRNA and piRNA analysis.
- CircRNA expression was assessed using RNaseR treatment and array hybridization.
- RT-qPCR validated reference gene stability.
Main Results:
- Trypsinization induced rapid alterations in the expression levels of multiple noncoding RNAs in hMSCs.
- These changes affected critical cellular pathways, indicating a significant biological response.
- Specific miRNAs, piRNAs, and circRNAs showed differential expression post-trypsinization.
Conclusions:
- Trypsinization is a potent stimulus that rapidly alters the noncoding RNA landscape in hMSCs.
- These noncoding RNA changes can impact cellular function and pathway activity.
- Careful consideration of trypsinization-induced noncoding RNA alterations is essential for robust experimental design and interpretation of results in hMSC research.
