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E z n a rna isolation columns

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The E.Z.N.A. RNA isolation columns are designed for high-quality RNA extraction from a variety of sample types. The columns facilitate the efficient capture, washing, and elution of RNA molecules, enabling researchers to obtain pure and concentrated RNA samples for downstream applications.

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Lab products found in correlation

Dna free dna removal kit, by Thermo Fisher Scientific (3 mentions) Trk lysis buffer, by Omega Bio-Tek (2 mentions) Qubit fluorometer, by Agilent Technologies (1 mentions) Tri reagent, by Molecular Research Center (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Sonic dismembrator model 100, by Thermo Fisher Scientific (1 mentions) Microbenrich kit, by Thermo Fisher Scientific (1 mentions) Rnaprotect cell reagent, by Qiagen (1 mentions) Lysozyme, by Merck Group (1 mentions) Rnaprotect bacteria reagent, by Qiagen (1 mentions) Proteinase k, by Qiagen (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions)

5 protocols using e z n a rna isolation columns

1

Splenic Myeloid Cell RNA Isolation

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Spleens were homogenized in TRI Reagent® (Molecular Research Center, Cincinnati, OH). Using the commercial product protocol, the upper aqueous layer following phase separation was mixed with an equal volume of 75% ethanol and transferred to E.Z.N.A.® RNA isolation columns (Omega Biotek, Norcross, GA). The manufacturer’s instructions were followed to complete tissue RNA isolation. RNA was isolated from approximately 7.5x104 splenic myeloid cells using the RNeasy® Plus Mini Kit (Qiagen, Hilden, Germany, Cat# 74134) according to the manufacturer’s instructions. RNA from cells or tissue was quantified using a Qubit Fluorometer and the RNA integrity was analyzed using an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA).
Povroznik J.M., Akhter H., Vance J.K., Annamanedi M., Dziadowicz S.A., Wang L., Divens A.M., Hu G, & Robinson C.M. (2023). Interleukin-27-dependent transcriptome signatures during neonatal sepsis. Frontiers in Immunology, 14, 1124140.
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2

Transcriptome Analysis of LAC Strain

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LAC was grown in LB with or without 30 mM itaconate to late exponential phase. Bacterial pellets were incubated in a cell wall lysis mixture (described above) at 37 °C for 45 min. TRK lysis buffer (Omega Bio-tek #R6834-02) and 70% ethanol were added, and samples were transferred to E.Z.N.A RNA isolation columns (Omega Bio-tek #R6834-02). RNA was isolated following the manufacturer’s instructions and treated with DNase using the DNAfree DNA removal kit (Fisher Scientific #AM1906). The RNA was precipitated with 0.1 volume 3 M sodium acetate (ThermoFisher #S209) and 3 volumes of 100% ethanol, recovered by centrifugation and washed with ice cold 70% ethanol. A ribosomal RNA-depleted cDNA library was prepared according to the manufacturer’s instructions using the Universal Prokaryotic RNA-Seq Prokaryotic AnyDeplete kit (NuGEN #0363-32) and sequenced with Illumina HiSeq. Raw base calls were converted to fatsq files using Bcl2fastqs. Filtered reads were aligned to the LAC_FPR3757 reference genome using STAR-Aligner v2.7.3a. The mapped reads were annotated for read groups and marked for duplicates using the Picard tools v2.22.3. The raw counts were quantified using Subreads:FeatureCounts v1.6.3 and processed for differential gene expression using DEseq2 in R v3.5.3.
Tomlinson K.L., Lung T.W., Dach F., Annavajhala M.K., Gabryszewski S.J., Groves R.A., Drikic M., Francoeur N.J., Sridhar S.H., Smith M.L., Khanal S., Britto C.J., Sebra R., Lewis I., Uhlemann A.C., Kahl B.C., Prince A.S, & Riquelme S.A. (2021). Staphylococcus aureus induces an itaconate-dominated immunometabolic response that drives biofilm formation. Nature Communications, 12, 1399.
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3

Isolation of Bacterial RNA from Infected Mouse Lungs

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Infected mouse lungs were excised and homogenized in PBS on ice. RNAprotect cell reagent (QIAGEN, 76526) was added to the lung homogenates, which were vortexed, incubated at RT for 10 min and pelleted. The pellets were resuspended in lysis buffer (as above) and incubated at RT for 5 min prior to sonication (Fisher Scientific sonic dismembrator model 100) on ice. TRK lysis buffer and 70% ethanol (v/v) were then added and the samples were transferred to E.Z.N.A. RNA isolation columns (Omega Bio-tek). Total RNA (mixed RNA from host and bacteria) was isolated following the manufacturer’s instructions and treated with DNase using the DNA-free DNA removal kit (Invitrogen, AM1906). The RNA was precipitated and bacterial RNA was enriched using the MICROBEnrich kit (Invitrogen, AM1901) according to the manufacturer’s instructions.
Wong Fok Lung T., Charytonowicz D., Beaumont K.G., Shah S.S., Sridhar S.H., Gorrie C.L., Mu A., Hofstaedter C.E., Varisco D., McConville T.H., Drikic M., Fowler B., Urso A., Shi W., Fucich D., Annavajhala M.K., Khan I.N., Oussenko I., Francoeur N., Smith M.L., Stockwell B.R., Lewis I.A., Hachani A., Baskota S.U., Uhlemann A.C., Ahn D., Ernst R.K., Howden B.P., Sebra R, & Prince A. (2022). Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection. Cell metabolism, 34(5), 761-774.e9.
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4

Bacterial RNA Isolation Protocol

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RNAprotect bacteria reagent (QIAGEN, 76506) was added to bacterial cultures, which were vortexed and incubated at room temperature (RT) for 10 min prior to centrifugation. Bacterial pellets were incubated in lysis buffer pH 8 (30 mM Tris (Corning, 46–030-CM), 1 mM EDTA (Thermo Fisher Scientific, 1861283), 15 mg/ml lysozyme (Sigma, L6876) and 200 μg/ml proteinase K (QIAGEN, 19131)) at RT for 10 min followed by the addition of TRK lysis buffer (Omega Bio-tek, R6834–02) and 70% ethanol (v/v). The samples were transferred to E.Z.N.A. RNA isolation columns (Omega Bio-tek). RNA was isolated following the manufacturer’s instructions and treated with DNase using the DNA-free DNA removal kit (Invitrogen, AM1906).
Wong Fok Lung T., Charytonowicz D., Beaumont K.G., Shah S.S., Sridhar S.H., Gorrie C.L., Mu A., Hofstaedter C.E., Varisco D., McConville T.H., Drikic M., Fowler B., Urso A., Shi W., Fucich D., Annavajhala M.K., Khan I.N., Oussenko I., Francoeur N., Smith M.L., Stockwell B.R., Lewis I.A., Hachani A., Baskota S.U., Uhlemann A.C., Ahn D., Ernst R.K., Howden B.P., Sebra R, & Prince A. (2022). Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection. Cell metabolism, 34(5), 761-774.e9.
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5

Inflammatory Cytokine Expression Analysis

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For assessment of inflammatory cytokines, total RNA was isolated using TRIzol reagent (Ambion by Life Technologies, Grand Island, NY) and E.Z.N.A. RNA Isolation Columns (Omega Bio-Tek, Norcross, GA). Reverse transcription was conducted using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Life Technologies, Grand Island, NY) according to manufacturer's instructions.
Real time RT-PCR was carried out with TaqMan gene expression assays. Targets include IL-6 (cat number: Mm.PT.51.12387735), IL-1β (cat number: Mm.PT.51.17212823), TNF-α (cat number: Mm.PT.51.16622079) and GAPDH (cat number: Mm.PT.39a.1) from Integrated DNA Technologies (Coralville, IA). GAPDH served as the endogenous housekeeping control. Reactions were performed in duplicate, and relative fold difference for each target gene was calculated using the 2−ΔΔCT method, where CT is the threshold concentration.
Vichaya E.G., Molkentine J.M., Vermeer D.W., Walker A.K., Feng R., Holder G., Luu K., Mason R.M., Saligan L., Heijnen C.J., Kavelaars A., Mason K.A., Lee J.H, & Dantzer R. (2015). Sickness behavior induced by cisplatin chemotherapy and radiotherapy in a murine head and neck cancer model is associated with altered mitochondrial gene expression. Behavioural brain research, 297, 241-250.
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