Rnazol rt

Manufactured by Zymo Research

RNAzol RT is a reagent used for the isolation and purification of total RNA from a variety of biological samples. It is a mono-phasic solution that includes phenol and guanidine thiocyanate, which facilitates the lysis of cells and the separation of RNA from DNA and proteins.

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Lab products found in correlation

Vero 76 cells, by American Type Culture Collection (1 mentions) Direct zol rna miniprep kit, by Zymo Research (1 mentions) Yeast trna, by Thermo Fisher Scientific (1 mentions) Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)

2 protocols using rnazol rt

Propagation and RNA Isolation of CHIKV

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CHIKV (strain 181/25 was obtained from BEI resources, Genbank no: EF452493.1) was propagated in Vero 76 cells (ATCC CRL-1587) grown in MEM-E with 10% FBS. After CPE was monitored, the cell supernatant was harvested and clarified with a low-speed centrifugation at 4,000 ×g for 20 min. The cell supernatant was stored at -80°C until use and viral RNA was isolated from the supernatants. RNA was isolated using RNAzol RT (MRC) with the Direct-zol RNA miniprep kit (Zymo Research) according to manufacturer’s instructions, with one additional wash step.

Wongsurawat T., Jenjaroenpun P., Taylor M.K., Lee J., Tolardo A.L., Parvathareddy J., Kandel S., Wadley T.D., Kaewnapan B., Athipanyasilp N., Skidmore A., Chung D., Chaimayo C., Whitt M., Kantakamalakul W., Sutthent R., Horthongkham N., Ussery D.W., Jonsson C.B, & Nookaew I. (2019). Rapid Sequencing of Multiple RNA Viruses in Their Native Form. Frontiers in Microbiology, 10, 260.

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VEEV Viral RNA Synthesis Assay

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VEEV viral RNA synthesis assay was adapted from Barton et al (Barton et al., 1991 (link)). Ten microliters of P15 fraction enriched for VEEV viral replicase complexes, which is equivalent of approximately 1.25 x 10⁶ infected cells, was combined with a same volume of a RNA synthesis mix (100 mM Tris-HCl pH 7.8, 100 mM KCl, 20 μg/mL act D, 20 mM DTT, 10 mM creatine phosphate, 50 μg/reaction creatine phosphokinase, 4 mM of ATP, GTP, and UTP, 20 μM CTP, 12 mM MgCl₂) on ice and 1 μL of SUPERase In RNase inhibitor (Ambion), 5 μg of yeast tRNA (Ambion), and 5 μCi of [α-³³P]-CTP (Perkin-Elmer) were added per reaction. After an incubation at 37 °C for 90 minutes, RNA was isolated from each reaction using RNAzol RT and RNA mini prep kit columns (Zymo Research) according to manufacturer’s instructions, with an additional wash step before elution. For compound addition, ML336 was added to reaction mixtures before incubation at 37 °C at the indicated concentrations. The final DMSO concentration was 0.25%.

Skidmore A.M., Adcock R.S., Jonsson C.B., Golden J.E, & Chung D.H. (2019). Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.. Antiviral research, 174, 104674.

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