Rneasy column kit

Manufactured by Qiagen

Sourced in United Kingdom

The RNeasy column kit is a laboratory equipment designed for the purification and extraction of RNA from various sample types. It utilizes a silica-based membrane to selectively bind and isolate RNA, allowing for efficient separation from other cellular components.

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RNeasy kit (11240 citations) RNeasy columns (885 citations) RNeasy Mini Kit columns (116 citations) RNeasy Mini Kit spin columns (8 citations) RNeasy Plus Mini kit columns (5 citations) RNeasy Mini Kit with on-column DNase digestion (4 citations) Mini Spin Columns of RNeasy Mini Kit (3 citations) RNeasy Plus Spin Column Kits (2 citations) RNeasy MinElute Cleanup Kit columns (2 citations) RNeasy MinElute spin column kit (2 citations)

14 protocols using rneasy column kit

RNA-seq Analysis of Transcriptome Changes

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Total RNA was extracted from cells of the indicated treatments using the RNeasy column kit (QIAGEN). Deep sequencing was performed using the Illumina HiSeq 2000 platform at the Génome Québec Innovation Centre (McGill University). The differential expression measurements were performed with DESeq2 v1.4.5. GEO accession number: GSE120268. Gene ontology analyses were conducted using Gene Ontology Consortium [37 (link)] and GSEA v2.1.0 (Gene Set Enrichment Analysis) [17 (link), 38 (link)]. For pharmacogenomics analysis, we used the PharmacoGx platform (version 1.1.5) to leverage the Connectivity Map data for drug repurposing analyses [14 (link)].

Goyette M.A., Cusseddu R., Elkholi I., Abu-Thuraia A., El-Hachem N., Haibe-Kains B., Gratton J.P, & Côté J.F. (2019). AXL knockdown gene signature reveals a drug repurposing opportunity for a class of antipsychotics to reduce growth and metastasis of triple-negative breast cancer. Oncotarget, 10(21), 2055-2067.

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RNA-Seq Analysis of Mouse Transcriptome

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RNA was extracted via RNeasy column kit (QIAGEN) following the manufacturer’s directions. Only samples with an RNA integrity number of 6 or higher as assessed by analysis on an Agilent Bioanalyzer were considered for RNA-Seq analysis. Sequencing was performed by GENEWIZ using a standard sequencing pipeline. In short, 30 million to 50 million read depth was achieved, and sequences were trimmed using Trimmomatic v.0.36. STAR aligner v.2.5.2b was used to map reads to the Ensembl mouse reference genome, after which unique gene hit counts of interexon regions were determined using FeatureCounts from the Subread package v.1.5.2. Differential expression analysis was conducted using DeSEQ2 in R v.3.6. A cutoff of ±1.5-fold change with an FDR ≤ 0.1 was used to identify differentially expressed genes. A full list of differentially expressed genes is available in the supplement (Supplemental Table 3). PCA and PERMANOVA were conducted in Python 3.7 using the Scitkitlearn learn package. GSEA was performed using the MSigDB immune signatures database (53 (link)).

Gurczynski S.J., Lipinski J.H., Strauss J., Alam S., Huffnagle G.B., Ranjan P., Kennedy L.H., Moore B.B, & O’Dwyer D.N. (2023). Horizontal transmission of gut microbiota attenuates mortality in lung fibrosis. JCI Insight, 9(1), e164572.

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Quantifying Il17a and Il23a mRNA

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The right lung was homogenized and total RNA purified using a RNeasy Column kit (Qiagen, Germantown, MD). RNA quantity and purity was determined using a small volume spectrophotometer (Nanodrop; Thermo Scientific, Waltham, MA). A total of 1 μg RNA was converted into cDNA using Super Script III First-strand amplification kit for qRT-PCR (Life Technologies, Grand Island, NY). Il17a and Il23a mRNA abundances were quantitated by real-time PCR (7300 Real-Time PCR Systems; Applied Biosystems, Carlsbad, CA) using intron-spanning primers (Kasahara et al., 2012 (link); Mathews et al., 2014b (link)) and SYBR-green detection. Il17a and Il23a were normalized to 36b4 expression using ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Brand J.D., Mathews J.A., Kasahara D.I., Wurmbrand A.P, & Shore S.A. (2016). Regulation of IL-17A Expression in Mice Following Subacute Ozone Exposure. Journal of immunotoxicology, 13(3), 428-438.

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RNA Extraction from Zebrafish Embryos

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Zebrafish embryos were injected with either PBCAB, NF-YA DN, GFP, or
antisense NF-YA DN mRNA as described above. At the desired timepoint,
embryos were collected into three biological replicates of 50–100
embryos per condition. Dead animals were counted, but excluded from RNA
extraction procedures. No other animals were excluded, and selection was not
blinded. Each sample was placed in 1mL of Trizol and frozen at
−80°C to help break up embryos. Once thawed, the embryos were
broken up by pipette and 250μL of chloroform was added to each sample
followed by vigorous shaking and a 3-minute incubation at room temperature.
The samples were then centrifuged at 12,000g for 15 minutes at 4°C
and th e aqueous phase was transferred to a new tube with 500mL of
isopropanol and 10μg of GlycoBlue (ThermoFisher Scientific). The
samples were vortexed, incubated at room temperature for 10 minutes, and
then centrifuged at 12,000g for 15 minutes at 4°C. The supernatant
was removed, and the pellet washed in 75% ethanol then centrifuged at 7,500g
for 5 minutes at room temperature. The supernatant was once again removed,
and the pellet was air-dried at room temperature for 10 minutes before
resuspension in 50μL of water. The samples were then further purified
and treated with DNase using the RNeasy Column kit (Qiagen) and eluted in
30μL of water.

Stanney W I.I.I., Ladam F., Donaldson I.J., Parsons T.J., Maehr R., Bobola N, & Sagerström C.G. (2019). Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish. Developmental biology, 459(2), 161-180.

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Quantification of PK2 and PKR Genes

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Total RNA was extracted from rat organotypic hippocampal slices using the RNeasy column kit (Qiagen, Milano, Italy). Purified RNA (1 μg) was used for cDNA synthesis using reverse transcriptase (Promega, Milan, Italy). cDNAs were amplified by real-time PCR (iCycler; Bio-Rad) using iQ SYBER Green Supermix (Bio-Rad) . Specific sense and antisense primers were synthesized (Biogen, Rome, Italy) to PCR amplify the rat PK2, PKR1 and PKR2 cDNA, according to the following sequences previously described (Giannini et al., 2009) : PK2, 5′-CAAGGACTCTCAGTGTGGA-3′ and 5′-AAAATGGAACTTTCCGAGTC-3′; PKR1, 5′-CGCACCGTCTCCCTCTAC-3 and 5′-GTTTGACACTTCATCCGCG-3′; PKR2, 5′CTCCGTCAACTACCTTCGTA-3′ and 5′-GAGGCGGTCTGGTAATTCA-3′.
β-Actin was used as internal standard. Results are presented as the Ct (cycle threshold) values of the specific gene of interest or as input copy number of the gene of interest per ng of total RNA. cDNA standards for each analyzed gene were generated, and serial dilutions ranging from 10 to 10 9 input copies were used as a standard curve in each PCR run.

Landucci E., Lattanzi R., Gerace E., Scartabelli T., Balboni G., Negri L, & Pellegrini-Giampietro D.E. (2016). Prokineticins are neuroprotective in models of cerebral ischemia and ischemic tolerance in vitro. Neuropharmacology, 108.

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Quantifying Gene Expression in Neutrophils

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Total RNA was isolated from neutrophils using TRI-reagent (Sigma, Poole, UK) followed by clean-up and DNase digest using RNeasy column kits (Qiagen). First-strand synthesis was performed with 1 μg of RNA (high-capacity cDNA kit, Applied Biosystems). Relative gene expression was determined by qPCR (ABI) and amplified in Sybr-green master mix (Applied Biosystems) with relevant primers from Qiagen. Gene expression levels were relative to β2 microglobulin using the 2^−CT method (42 (link)).

Pocock J.M., Storisteanu D.M., Reeves M.B., Juss J.K., Wills M.R., Cowburn A.S, & Chilvers E.R. (2017). Human Cytomegalovirus Delays Neutrophil Apoptosis and Stimulates the Release of a Prosurvival Secretome. Frontiers in Immunology, 8, 1185.

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Gene Expression Analysis of Skin Samples

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Total RNA was isolated from skin using TRI-reagent (Sigma), followed by RNA clean-up and DNase digest using RNeasy column kits (Qiagen, Manchester, UK). First-strand synthesis was performed with 1 μg of total RNA using a high-capacity cDNA kit (Applied Biosystems, Paisley, UK) according to the manufacturer’s instructions. Relative gene expression was determined by qPCR (ABI system, Applied Biosystems) and amplified in Sybr-green master mix (Roche) using relevant primers from Qiagen. Deletion efficiency was characterised for NOS2 in the K14cre+ mouse using DNA isolated from skin samples using TRIzol/DNeasy columns (Qiagen, Manchester. UK). The PCR primers and Taqman probe were designed in-house and synthesised by Sigma (Gillingham UK) fwd 5’-TCCAGAATCCCTGGACAAG rev 5’-TGGTGAAGAGTGTCATGCAA, probe 5’-FAM-TGTGACATCGACCCGTCCACA.

Cowburn A.S., Macias D., Summers C., Chilvers E.R, & Johnson R.S. (2017). Cardiovascular adaptation to hypoxia and the role of peripheral resistance. eLife, 6, e28755.

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Quantitative RNA Expression Analysis

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Total RNA was isolated from tissues using TRI-reagent (Sigma) followed by RNA clean-up and DNase digest using RNeasy column kits (Qiagen, Venlo, the Netherlands). First-strand synthesis was performed with 1 μg of total RNA using Superscript IV (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Relative gene expression was determined using quantitative (q)PCR (One-Step Plus Real-Time PCR System; Life Technologies, Waltham, MA, USA) and was amplified in SYBR-Green master mix (Roche, Basel, Switzerland) and relevant primers from Qiagen. Relative gene-expression levels were corrected to housekeeping genes β-actin and B₂M.

Macias D., Moore S., Crosby A., Southwood M., Du X., Tan H., Xie S., Vassallo A., Wood A.J., Wallace E.M, & Cowburn A.S. (2021). Targeting HIF2α-ARNT hetero-dimerisation as a novel therapeutic strategy for pulmonary arterial hypertension. The European Respiratory Journal, 57(3), 1902061.

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UV-Crosslinking Liver Proteome-RNA Interactome

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100mg liver powder, on dry ice, was cross linked with UV (254 nm, 400 mJ/cm²) three times. AG dynabeads were washed twice in lysis buffer (50 mM Tris−HCl (7.4), 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxcholate) and incubated with 10 μg antibody rotating at 4°C for 1 h then washed once in high salt buffer (50 mM Tris–HCl (7.4), 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxcholate) and twice in lysis buffer. Liver powder was resuspended in 1 ml lysis buffer (+Proteinase inhibitors & RNase inhibitors) and lysed using a TissueLyser II Homogenizer (Qiagen). 16 mg of protein was incubated at 37°C for 3 min with 10μl 1/1000 RNaseI (in lysis buffer) and 2 μl Turbo DNase then 3 min on ice. Samples were span at 18 000g for 10 min and the supernatant mixed with antibody bound beads and incubated at 4°C rotating for 1 h. Beads were washed twice with high salt buffer. 1/10 of beads were kept to assess immunoprecipitation efficiency and the rest treated with 50 μg PK to release RNA. RNA was purified using RNeasy kit column (Qiagen) and quantified using a nanodrop.

Jobbins A.M., Haberman N., Artigas N., Amourda C., Paterson H.A., Yu S., Blackford S.J., Montoya A., Dore M., Wang Y.F., Sardini A., Cebola I., Zuber J., Rashid S.T., Lenhard B, & Vernia S. (2022). Dysregulated RNA polyadenylation contributes to metabolic impairment in non-alcoholic fatty liver disease. Nucleic Acids Research, 50(6), 3379-3393.

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Quantifying P2Y4 Receptor mRNA in Transfected Cells

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Total mRNAs were extracted from different clonal transfected 1321N1 cell cultures in TRIzol reagent followed by purification with RNeasy kit column (Qiagen, Antwerp, Belgium). mRNA was reverse transcribed using random hexamers and Superscript II Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, United States). qRT-PCR amplification mixtures contained 10 ng template cDNA and the specific P2Y₄ primers C and D (Table 1). Reactions were run on a 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, United States) with an annealing temperature of 60°C for primers C and D. qRT-PCR data were expressed as Ct obtained for P2Y₄ mRNA in clonal transfected 1321 cells.

Horckmans M., Diaz Villamil E., Verdier C., Laurell H., Ruidavets J.B., De Roeck L., Combes G., Martinez L.O, & Communi D. (2022). Loss-of-function N178T variant of the human P2Y4 receptor is associated with decreased severity of coronary artery disease and improved glucose homeostasis. Frontiers in Pharmacology, 13, 1049696.

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