Sureprep rna cleanup and concentration kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SurePrep RNA Cleanup and Concentration Kit is a laboratory product designed to purify and concentrate RNA samples. It removes contaminants and impurities from RNA preparations, allowing for the recovery of high-quality RNA suitable for downstream applications.

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RNA cleanup kit (3 citations)

5 protocols using sureprep rna cleanup and concentration kit

RNA Extraction and Purification Protocol

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Total RNA was extracted from at least 200,000 cells collected at the end of dead cell removal step, using an RNeasy Plus mini kit according to the manufacturer’s instructions (Germantown). Total RNA was eluted in 30 μl of RNA-free water. Genomic DNA was removed by using deoxyribonuclease I (Thermo Fisher Scientific), and the purified RNA was recovered using the SurePrep RNA Cleanup and Concentration Kit, according to the manufacturer’s instructions (Fisher Scientific). The genomic DNA removal and cleanup step was repeated a second time. The purity of the finally obtained total RNA was evaluated using Bioanalyzer. Sequencing was performed on NovaSeq 6000 at the Center for Genomics and Bioinformatics, Indiana University, School of Medicine.

Szymczak F., Alvelos M.I., Marín-Cañas S., Castela Â., Demine S., Colli M.L., Op de Beeck A., Thomaidou S., Marselli L., Zaldumbide A., Marchetti P, & Eizirik D.L. (2022). Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic β cells. Science Advances, 8(37), eabn5732.

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RNA Isolation and qRT-PCR Quantification

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RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol for chloroform extraction. Samples were additionally processed with SurePrep RNA Cleanup and Concentration kit (Fisher BioReagents, Fair Lawn, NJ, USA). The polysome profile RNA isolation included 10 μg glycogen in each sample. A total of 200 ng RNA was reverse‐transcribed using QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA, USA). qRT–PCR was performed in technical duplicate using KAPA SYBR FAST qPCR Master Mix on a LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA). Target gene mRNA was normalized to the housekeeping gene cdc‐42, except for instances of polysome analysis, in which compared samples were normalized to the housekeeping gene act‐1, which we have found does not change among total and translated pools between N2 and ifg‐1, nor before or after exposure to tunicamycin. Changes in gene expression levels were analyzed by using the 2−ΔΔCT method. Primer sequences are provided in Table S8 (Supporting information).

Howard A.C., Rollins J., Snow S., Castor S, & Rogers A.N. (2016). Reducing translation through eIF4G/IFG‐1 improves survival under ER stress that depends on heat shock factor HSF‐1 in Caenorhabditis elegans. Aging Cell, 15(6), 1027-1038.

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RNA Extraction and qRT-PCR Analysis

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The total RNA was extracted from 2D monolayer cell cultures or CTCs from 4D model with Isol-RNA lysis reagent (5 PRIME) followed by the SurePrep RNA cleanup and concentration kit (Fisher Scientific, Pittsburgh, PA, USA). RNA was treated with the Ambion DNA-free kit (Applied Biosystems, Carlsbad, CA, USA) as per the manufacturer’s instructions. The cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) with 500 ng of total RNA, and the real-time PCR assay was prepared with SensiFast SYBR green reagent (Bioline USA Inc., Taunton, MA, USA). The relative gene expression of target genes was determined against the reference genes L32 (16 (link)) and 18s (17 ) for mice and humans, respectively. The primers for human and mice target genes were designed using Primer 3.0 online software (SimGene.com) (18 (link)) (Table 1). We calculated the relative fold of gene expression using 2−(ΔΔCt) formula.

Mishra D.K., Scott K.L., Wardwell-Ozgo J.M., Thrall M.J, & Kim M.P. (2014). Circulating Tumor Cells from 4D Model Have Less ITGB4 Expression. The Journal of surgical research, 193(2), 745-753.

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Comprehensive RNA Extraction and Sequencing

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RNAs were prepared as described (Sallet et al., 2013) . Briefly, total plant and bacterial RNAs were extracted using the MirVana™ miRNA isolation kit (Ambion), treated with the Turbo DNAse (Ambion) and purified with the SurePrep™ RNA cleanup and concentration kit (Fisher BioReagents™). A ribosomal RNA depletion was performed, using an oligocapture strategy based on the Plant Ribominus kit (Invitrogen), with an oligonucleotide set specifically targeting M. truncatula and S. meliloti rRNAs and the highly abundant S. meliloti tRNA-Alanine. Samples were then once more treated with the Turbo DNAse (Ambion) and purified on Zymo Research RNA Clean & Concentrator TM-5 columns (Proteigene). RNA sample quality was assessed using the RNA 6000 Nano kit and a Bioanalyser (Agilent).
RNA-seq libraries were prepared according to Illumina's protocols and the Illumina TruSeq Stranded mRNA sample prep kit. Oriented paired-ended RNA sequencing (2 x 150 bp) was performed on Illumina HiSeq3000.

Sauviac L., Rémy A., Huault E., Dalmasso M., Kazmierczak T., Jardinaud M.F., Legrand L., Moreau C., Ruiz B., Cazalé A.C., Valière S., Gourion B., Dupont L., Gruber V., Boncompagni E., Meilhoc E., Frendo P., Frugier F, & Bruand C. (2022). A dual legume-rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses. Plant, cell & environment, 45(10).

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Plant Ribosomal RNA Depletion for RNA-Seq

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A RiboMinus™ Plant Kit for RNA-Seq (Invitrogen) was used for the removal of plant ribosomal RNA (rRNA) from total RNA in accordance with the manufacturer's manual. Then, the rRNA-free RNA fraction was concentrated by precipitation with 2 µL of GlycoBlue™ Coprecipitant (15 µg/µL; Invitrogen), 1/10 V of 3 M sodium acetate, pH 5.5 (Invitrogen), and 3 V of 96% ethanol. RNA precipitate was washed with 70% ethanol and resuspended with 40 µL of RNAse free water. Residual oligonucleotides were removed with DNAse I (Zymo Research, Irvine, CA, USA) and RNA was purified using a Clean & Concentrator™ kit (Zymo Research) in accordance with the manufacturer's instructions. The RNA concentration was measured with Qubit 2.0. To obtain a sufficient amount of mRNA for transcriptome analysis and real-time PCR, a single round of RNA amplification was performed with a MessageAmp II Bacteria kit (Invitrogen). Only plant mRNA was amplified because the polyadenylation step was omitted. The cDNA templates were then removed using DNAse I (Zymo Research) and amplified RNA purification was carried out with a SurePrep RNA Cleanup and Concentration Kit (Fisher BioReagents, Pittsburgh, PA, USA) according to the manufacturer's instructions.

Kusakin P.G., Serova T.A., Gogoleva N., Gogolev Y., & Tsyganov V.E. (2021). Laser Microdissection of Pisum sativum L. Nodules Followed by RNA-Seq Analysis Revealed Crucial Transcriptomic Changes during Infected Cell Differentiation. Agronomy, 11(12), 2504-2504.

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