RNA graanulite uurimus inimese neuroblastoomi SH-SY5Y rakuliinis

We aimed to establish a protocol for the purification of neuronal RNA granules from all-trans retinoic acid differentiated human neuroblastoma cell line SH-SY5Y. Different bio-chemical purification methods were tested for obtaining a homogenous preparation of RNA granules for later structural analysis by cross-linking coupled mass-spectrometry or single-particle cryo-electron microscopy. The major problem encountered was a significant amount of copurifying cellular glycogen granules in the RNA granule preparations obtained by a combination of velocity sedimentation and size-exclusion chromatography. However, a ma-jority of the contaminating glycogen could be removed from the RNA granule preparations using a maltose-binding protein (MBP) or artificial FLX protein based Ni-Sepharose or Flag-resin affinity chromatography. Besides, changes in the expression levels of mRNAs im-portant for neuronal differentiation and synaptic function upon SH-SY5Y differentiation were analyzed. A western blot analysis revealed the presence of key RNA granule compo-nents previously identified in rat cortical RNA granules (e.g. CAPRIN-1, G3BP-1, and G3BP-2) in the SH-SY5Y derived RNA granules. In estonian: Käesoleva bakalaureusetöö eesmärgiks oli välja töötada meetodid inimese luuüdikasvajast pärit SH-SY5Y rakuliini kasutamiseks in vitro mudelsüsteemina neuronaalsete RNA graanulite ekspressiooniks ja puhastamiseks. Töö käigus testiti erinevaid biokeemilise puhastamise meetodeid võimalikult homogeense RNA graanulite preparatsiooni valmistamiseks retinoolhappe toimel differentseeritud SH-SY5Y rakkudest hilisemaks RNA graanulite struktuuri analüüsiks ristsidumise massispektromeetria või krüo- elektronmikroskoopia abil. Peamiseks raskuseks osutus töös glükogeeni graanulite kaasapuhastumine RNA graanulitega. RNA graanulite esialgse preparaadi täiendav puhastamine maltoosi siduva valgu või tehisvalgu FLX afiinsusresinil (Ni-sefaroos või Flag, vastavalt) võimaldas siiski kontamineerivat glükogeeni edukalt eemaldada. Töö käigus analüüsiti lisaks retinoolhappe poolt indutseeritud differentseerumisega kaasnevaid muutusi neuronaalsete mRNA-de ekspressioonitasemes SH-SY5Y rakkudes. Western blot analüüs tuvastas roti ajukoorest eraldatud RNA graanulitele omaste oluliste RNA graanuli funktsiooni regulerivate valkude nagu Caprin-1, G3BP-1 ja G3BP-2 olemasolu SH-SY5Y rakkudest eraldatud RNA graanulites

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data