High-Throughput Spectroscopic Analysis of mRNA Capping Level.

Eukaryotic mRNAs are characterized by terminal 5' cap structures and 3' polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.

Tetracycline-mediated fluorescence correlates with passage number and ß-galactosidase activity in HeLa and HEK293T cells.

We report data consistent with tetracycline-mediated fluorescence having the potential to be an effective marker of senescence in immortalised cells. HeLa cells that had previously undergone more than 20 passages were transiently transfected with a plasmid encoding a novel tetracycline-inducible transgene featuring an open reading frame for green fluorescent protein. While characterising the performance of this plasmid and transfection procedure, HeLa cell fluorescence was observed to result from incubating cells with media containing 2 µg/ml tetracycline alone, without plasmid or transfection reagent. To investigate this phenomenon further, HeLa and HEK293T cells were purchased from a tissue culture collection and after cultivation over 4-23 passages, incubated with media containing 2 µg/ml tetracycline. For both cell lines, tetracycline-mediated fluorescence increase correlated with passage number increase. This effect in HeLa and HEK293T cells was also borne out by expression of ß-galactosidase activity, an imperfect but widely used marker of cellular senescence. These data suggest tetracycline may have utility as a marker of cellular senescence in immortal cells and can inform future investigation and validation of this previously unreported application for this reagent.