Rna clean concentrator 5 column

Manufactured by Zymo Research

Sourced in United States

The RNA Clean & Concentrator-5 column is a laboratory equipment designed for the purification and concentration of RNA samples. It utilizes a silica-based membrane to selectively bind RNA, allowing for the removal of contaminants and the concentration of the target RNA sample.

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30 protocols using rna clean concentrator 5 column

Circular RNA Extraction and Purification

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The circularization of the adaptor-ligated RNA (RNA:adaptor) was carried out at 25 °C for 2h, in a total volume of 20 µL containing 10 U of T4 RNA Ligase 1 (NEB, cat n° M0204L), 1X T4 RNA ligase buffer, 20% PEG8000, 50 µM ATP. The reaction was then incubated at 37 °C for 1 h with 20 U of RNase R (Lucigen, cat n°RNR07250), to remove all the undesired products (i.e linear RNA or concatemer product). Circular RNA was purified through RNA Clean & Concentrator™-5 column (Zymo research, Cat. n° R1013) and quantified using Qubit™ RNA HS Assay Kit (Thermo Fisher, Catalog number: Q32852).

Piano A.D., Barbieri R., Schmid M., Brocchieri L., Tornaletti S., Firrito C., Minati L., Bernabò P., Signoria I., Lauria F., Gillingwater T.H., Viero G., & Clamer M. (2020). Phospho-RNA sequencing with CircAID-p-seq.

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RNA Fragmentation and Reverse Transcription

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Total RNA was extracted from parasite lysates without any chemical reagent modification. In a 20-μL reaction, ~2 μg of total RNA was fragmented using RNA fragmentation reagents (Ambion) for the proper time at 70°C. After the end of the experiment, 10× stop solution was added to each sample, and the samples were immediately placed on ice. Next, fragmented RNA was purified with a Zymo RNA Clean & Concentrator-5 column. The purified fragmented RNA was reverse-transcribed by primer extension with gene-specific colour-coded fluorescently FAM-labelled oligonucleotides (5’-ACCCTAACATCAAAAGCTGATAGG-3’), as described above. The length of every fragment was assessed using an ABI 3730XL DNA Sequencer (Applied Biosystems). The results were shown by GeneMarker (V2.7.0, SoftGenetics, LLC.).

Qi Y., Zhang Y., Zheng G., Chen B., Zhang M., Li J., Peng T., Huang J, & Wang X. (2021). In Vivo and In Vitro Genome-Wide Profiling of RNA Secondary Structures Reveals Key Regulatory Features in Plasmodium falciparum. Frontiers in Cellular and Infection Microbiology, 11, 673966.

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RHV Genomic RNA Extraction Protocol

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For the extraction of RHV genomic RNA, 250 µl supernatant from infected cell cultures, density gradient fractions, or rodent serum samples (25 µl diluted in 225 µl PBS) were added to a 2‐ml Phasemaker tube (Thermo Fisher Scientific) and mixed with 750 µl TRIzol LS Reagent (Thermo Fisher Scientific). After addition of 200 µl chloroform, vigorous shaking for 15 s, 3 min incubation at room temperature, and centrifugation at 12,000g for 15 min at 4°C, the aqueous phase was mixed with 450 μl anhydrous ethanol and transferred to an RNA Clean & Concentrator‐5 column (Zymo Research) for downstream RNA purification and concentration. The complete RHV open reading frame (ORF) was amplified and subjected to deep‐sequencing analysis as previously described.^[¹⁴^] RHV RNA was quantified by one‐step TaqMan reverse‐transcription quantitative PCR, as previously described.^[¹⁵^] Details on isopycnic centrifugation and reverse‐transcription quantitative PCR primers are specified in Text S1.

Wolfisberg R., Thorselius C.E., Salinas E., Elrod E., Trivedi S., Nielsen L., Fahnøe U., Kapoor A., Grakoui A., Rice C.M., Bukh J., Holmbeck K, & Scheel T.K. (2022). Neutralization and receptor use of infectious culture–derived rat hepacivirus as a model for HCV. Hepatology (Baltimore, Md.), 76(5), 1506-1519.

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In Vitro MRPS21 Transcript Folding

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dsDNA corresponding to the human MRPS21 sequences shown below were in vitro transcribed, mixed, and folded by denaturing at 95°C followed by a brief incubation at 37°C in 350 mM sodium cacodylate buffer and 6 mM MgCl₂. 10% DMS (v/v) was added, and the sample was incubated for 10 min at 37°C. The reaction was stopped by placing on ice and adding BME to 30% final volume. The RNA was then purified by RNA Clean & Concentrator-5 column (Zymo), and the small RNA fraction was collected and prepared for sequencing as described in the genome-wide strategy above.

Zubradt M., Gupta P., Persad S., Lambowitz A.M., Weissman J.S, & Rouskin S. (2016). DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nature methods, 14(1), 75-82.

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RHV Genomic RNA Extraction and Sequencing

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For the extraction of RHV genomic RNA, 250 µL supernatant from infected cell cultures, density gradient fractions, or rodent serum samples (25 µL diluted in 225 µL PBS) were added to a 2 mL Phasemaker tube (Thermo Fisher) and mixed with 750 µL TRIzol LS Reagent (Thermo Fisher). After addition of 200 µL chloroform, vigorous shaking for 15 s, 3 min incubation at room temperature, and centrifugation at 12,000 g for 15 min at 4 °C, the aqueous phase was mixed with 450 μL anhydrous ethanol and transferred to an RNA Clean & Concentrator-5 column (Zymo Research) for downstream RNA purification and concentration. The complete RHV open reading frame (ORF) was amplified and subjected to deep sequencing analysis as previously described (14 (link)). RHV RNA was quantified by one-step TaqMan reverse transcription quantitative PCR (RT-qPCR), as previously described (15 ). Details on isopycnic centrifugation and RT-qPCR primers are specified in Supporting Text S1.

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Circular RNA purification protocol

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The circularization of the adaptor-ligated RNA (RNA:adaptor) was carried out at 25°C for 2 h, in a total volume of 20 μl containing 10 U of T4 RNA Ligase 1 (NEB, cat. no. M0204L), 1× T4 RNA ligase buffer, 20% PEG8000, 50 μM ATP. The reaction was then incubated at 37°C for 1 h with 20 U of RNase R (Lucigen, cat. no. RNR07250), to remove undesired products (i.e. linear RNA or concatemer product). Circular RNA was purified by using RNA Clean & Concentrator™-5 column (Zymo research, cat. no. R1013) and quantified using Qubit™ RNA HS Assay Kit (Thermo Fisher, cat. no. Q32852).

Del Piano A., Kecman T., Schmid M., Barbieri R., Brocchieri L., Tornaletti S., Firrito C., Minati L., Bernabo P., Signoria I., Lauria F., Gillingwater T.H., Viero G, & Clamer M. (2021). Phospho-RNA sequencing with circAID-p-seq. Nucleic Acids Research, 50(4), e23.

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RNA Depletion and Nanopore Sequencing Workflow

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Total RNA was first depleted using the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Illumina). RNA was then purified and concentrated on a RNA Clean Concentrator™-5 column (Zymo Research, Irvine, CA, USA). cDNA libraries were performed from a mix of 50 ng RNA and 0.5 ng Spike-in RNA Variant Control Mix E2 (Lexogen) according to the Oxford Nanopore Technologies (Oxford Nanopore Technologies Ltd, Oxford, UK) protocol “DNA-PCR Sequencing” with a 14 cycles PCR (8 minutes for elongation time). ONT adapters were ligated to 650 ng of cDNA.

Sessegolo C., Cruaud C., Da Silva C., Cologne A., Dubarry M., Derrien T., Lacroix V, & Aury J.M. (2019). Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules. Scientific Reports, 9, 14908.

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RNA Extraction from Bacterial Cells

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For RNA‐seq analysis, cells were grown in LB medium at 37°C and 220 rpm and harvested in the mid‐ to late‐exponential phase or stationary phase. Cell pellets were re‐suspended in 200 µl of TE buffer, after which 1% (final) SDS and 0.25 g of glass beads were added. After two times bead beating for 1 min (1 min on ice in between), 1 ml of ice‐cold TRIzol was added and samples were vortexed and incubated for 20 min at 70°C to fully lyse the cells. 200 µl of chloroform was then added, followed by vortexing for 15 s and incubation for 2 min at RT. After centrifugation (12 500 g, 15 min, 4°C), the upper aqueous phase containing RNA was transferred to a clean tube and mixed with two volumes of 100% ethanol. RNA was purified using an RNA Clean & Concentrator™‐5 column (Zymo Research, cat. R1015) according to manufacturer instructions. RNA concentrations were measured using a NanoDrop ND‐1000 (Thermo Fisher Scientific). RNA quality was assessed by gel electrophoresis. RNA samples were stored at −80°C.

van Tilburg A.Y., Warmer P., van Heel A.J., Sauer U, & Kuipers O.P. (2021). Membrane composition and organization of Bacillus subtilis 168 and its genome‐reduced derivative miniBacillus PG10. Microbial Biotechnology, 15(5), 1633-1651.

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RNA SHAPE and DMS Probing

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For in vitro SHAPE probing of RNA, NAI was added to a final concentration of 50 mM and samples were incubated at 37°C for 10 min. A negative control reaction was also prepared, by adding an equal amount of DMSO. For DMS probing, dimethyl sulfate (Sigma Aldrich, cat. D186309) from a 1:6 dilution in 100% ethanol was added at a final concentration of 150 mM and samples were incubated at 37°C for 2 min. Reactions were then quenched by the addition of 1 volume DTT 1 M and then purified on an RNA Clean & Concentrator-5 column (Zymo Research, cat. R1013).

Manfredonia I., Nithin C., Ponce-Salvatierra A., Ghosh P., Wirecki T.K., Marinus T., Ogando N.S., Snijder E.J., van Hemert M.J., Bujnicki J.M, & Incarnato D. (2020). Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements. Nucleic Acids Research, 48(22), 12436-12452.

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RNA Extraction and Purification Protocol

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After samples have been collected in 1 ml QIAzol, 200 μl of chloroform was added, followed by vigorous vortexing for 15 s. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12 500 × g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 ml tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column (Zymo Research, cat. R1015) and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Besides samples treated with I6, all other samples appeared to be perfectly intact. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase (ThermoFisher Scientific, cat. AM2239) at 37°C for 20 min.

Marinus T., Fessler A.B., Ogle C.A, & Incarnato D. (2021). A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy. Nucleic Acids Research, 49(6), e34.

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