RESUMEN
Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.
RESUMEN
BACKGROUND: The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure. METHODS: For this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression. RESULTS: The biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice. CONCLUSIONS: We have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.