Total yeast rna

Manufactured by Merck Group

Sourced in United Kingdom

Total yeast RNA is a laboratory product designed for use in various scientific applications. It provides a source of RNA extracted from the cells of the yeast Saccharomyces cerevisiae. The core function of this product is to serve as a raw material for downstream RNA-based experiments and analyses, such as gene expression studies, RNA sequencing, and other molecular biology techniques.

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Lab products found in correlation

Syto13, by Thermo Fisher Scientific (1 mentions) Polyadenylic acid, by Merck Group (1 mentions) Rnase free water, by Santa Cruz Biotechnology (1 mentions) Polyuridylic acid, by Merck Group (1 mentions) Imagequant software, by GE Healthcare (1 mentions) A1r eclipse ti, by Nikon (1 mentions)

Close spelling variants of this product

Yeast RNA

5 protocols using total yeast rna

Biomolecular Interactions of Peptides and RNA

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All the peptides used ([RGRGG]₅, [KGKGG]₅, [KGYGG]_5, RGG-3 domain of FUS) were purchased from Genscript USA Inc. (NJ, USA). All peptides contained a C-terminus cysteine for site-specific peptide labeling. Peptide stock solutions were prepared in RNase-free water (Santa Cruz Biotechnology) with 50 mM dithiothreitol (DTT). Polyuridylic acid [poly(rU); Sigma-Aldrich; molecular weight = 600–1000 kDa], Polyadenylic acid [poly(rA); Sigma-Aldrich; molecular weight = 100–500 kDa], Poly(phosphate) [poly(P) p100, medium-chain; Kerafast Inc; molecular weight = 11.5 kDa], custom-synthesized RNA oligomer poly(rU)-rU40 [40 nucleobases; Integrated DNA Technologies (IDT); molecular weight = 12185 Da], yeast total RNA (Sigma-Aldrich)] and FAM-labeled RNA oligomer-rU10 [Integrated DNA Technologies (IDT)] were reconstituted in RNase-free water. The concentration of all RNAs was calculated from their respective measured absorbance at 260 nm in a UV–Vis spectrophotometer (Nanodrop oneC). Both RNA and peptide stock solutions were stored at −20 °C. Before sample preparation for experiments, the RNA [poly(rU), poly(rA), and rU40] stock solutions were checked for any aggregates using bright-field microscopy. Nucleic acid staining dye SYTO13 was purchased from Thermo Fisher Scientific Inc.

Kaur T., Raju M., Alshareedah I., Davis R.B., Potoyan D.A, & Banerjee P.R. (2021). Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies. Nature Communications, 12, 872.

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Structural Probing of Stem II RNA

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Structural probing was carried out following methods described by Wroblewska and Olejniczak [49 (link)]. Each reaction contained 9 ng of stem II RNA and 1 μg of yeast total RNA (Sigma-Aldrich, St. Louis, MO) diluted to 5 μl with RNase-free water. 5x buffer (2μL; 250 mM Tris pH 7.5, 10 mM EDTA) was then added. T1 RNase (3 μl; 0.03 U/μl) was added to each sample and incubated at 37°C for 3 min. T1 Stop buffer (10 μL; 8M urea, 40 mM EDTA, 50% deionized formamide, 0.02% w/v bromophenol blue, 0.02% w/v xylene cyanol) was then added. The U2 stem II T1 RNase ladder showing cleavage at every guanine was obtained from 9 μg of stem II RNA and 1 μg of yeast total RNA in 50 mM sodium acetate pH 4.3, 7M Urea buffer by digestion with 1U of T1 RNase at 55°C for 10 min before stopping the reaction by addition of equal volume of Stop buffer. An RNA ladder was produced by incubating 9 ng of stem II RNA and 1 μg of yeast total RNA with 50 mM sodium acetate pH 9.7 for 2 min at 90°C before adding an equal volume of Stop buffer. Samples and ladders were then analyzed by denaturing 10% PAGE and the gel was dried before the bands were imaged using a phosphorscreen. Band intensities were quantified using ImageQuant software (GE Healthcare Life Sciences, Pittsburgh, PA).

van der Feltz C., DeHaven A.C, & Hoskins A.A. (2017). Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II. Journal of molecular biology, 430(4), 524-536.

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Measuring ATP Hydrolysis of DDX5

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In vitro ATP hydrolysis assays were performed using an enzyme‐coupled absorbance assay.36 ATP hydrolysis rate was measured using 400 nM of purified, recombinant MBP‐DDX5‐GST, or mutants in the presence and absence of 250 ug/mL of total yeast RNA (Sigma‐Aldrich, St. Louis, MO). k_obs values were calculated using the formula: V₀ = (A₃₄₀/min × 2.5)/(6.22 × 10⁻³μm), where k_obs(min⁻¹) = V₀/protein concentration. DDX5 fused to an N‐terminal MBP and C‐terminal GST tag (MBP‐DDX5‐GST) was expressed in Escherichia coli and purified by affinity chromatography. DDX5 mutants were purified by the same method (N = 3; error bars represent SD).

Zhang H., Xing Z., Mani S.K., Bancel B., Durantel D., Zoulim F., Tran E.J., Merle P, & Andrisani O. (2016). RNA helicase DEAD box protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus infection and hepatocarcinogenesis. Hepatology (Baltimore, Md.), 64(4), 1033-1048.

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ATP Hydrolysis Assay for DDX5

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In vitro ATP hydrolysis assays were performed using an enzyme-coupled absorbance assay (36 (link)). ATP hydrolysis rate was measured using 400 nM purified, recombinant MBP-DDX5-GST or mutants in the presence and absence of 250 ug/ml total yeast RNA (Sigma). k_obs values were calculated using the formula: V₀ = (A₃₄₀/min × 2.5)/(6.22 × 10⁻³μM), where k_obs(min⁻¹) = V₀/protein concentration. DDX5 fused to an N-terminal MBP and C-terminal GST tag (MBP-DDX5-GST) was expressed in E.coli and purified by affinity chromatography. DDX5 mutants were purified by the same method. N=3, error bars represent standard deviation.

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Visualizing TDP-43 Phase Separation

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Total yeast RNA (Sigma, Gillingham, UK; Cat N° 10109223001) was prepared in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 7.0) or phosphate buffer (50 mM, pH 7.5). For fluorescence microscopy, we spiked the phase-separating mixture with TDP-43 labeled with Alexa Fluor™ 488 at 1:100 ratio. We incubated the samples for 5 min (unless otherwise stated) in incubation chambers (grace biolabs, 666,106 JTR12R-A2-1.0) sealed with 1.5# coverslips coated with PEG silane. Samples were visualized by fluorescence microscopy (Nikon A1R Eclipse Ti, Tokyo, Japan) using a 488 laser and Apo 60X objective. Images were taken in approximately the middle of the incubation chamber to avoid wetting effects on the coverslips.

Pakravan D., Michiels E., Bratek-Skicki A., De Decker M., Van Lindt J., Alsteens D., Derclaye S., Van Damme P., Schymkowitz J., Rousseau F., Tompa P, & Van Den Bosch L. (2021). Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation. Biomolecules, 11(4), 548.

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