Rneasy lysis buffer rlt

Manufactured by Qiagen

Sourced in Germany

RNeasy lysis buffer RLT is a laboratory reagent used for the lysis and disruption of cells and tissues to facilitate the extraction and purification of RNA. It is a key component of the RNeasy kit series offered by Qiagen for RNA isolation and purification.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (3 mentions) Rneasy kit, by Qiagen (2 mentions) Nextseq 500 instrument, by Illumina (2 mentions) Nextflex dna barcode, by PSC Biotech (2 mentions) High sensitivity dna bioanalyzer, by Agilent Technologies (2 mentions) Kapa rna hyperprep kit with riboerase human mouse rat hmr, by Roche (2 mentions) Tissuelyser 2, by Qiagen (2 mentions) Biosprint 96, by Qiagen (2 mentions) Nucleomag vet kit, by Macherey-Nagel (2 mentions) Facsaria 3 flow cytometer, by BD (1 mentions) Facsaria fusion, by BD (1 mentions) Cryostor cs10, by Merck Group (1 mentions) Viia7 qpcr instrument, by Thermo Fisher Scientific (1 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Rnasin plus rnase inhibitor, by Promega (1 mentions) Qiacube, by Qiagen (1 mentions) Dnase 1, by Qiagen (1 mentions) Nucleospin rna xs kit, by Takara Bio (1 mentions) Nanodrop, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

RLT buffer (1835 citations) RLT lysis buffer (402 citations) Lysis buffer (117 citations) RNeasy lysis buffer (46 citations) RNeasy RLT buffer (8 citations) Buffer RLT lysis buffer (5 citations)

11 protocols using rneasy lysis buffer rlt

FACS Analysis of DENV2 Infection

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Fluorescence-activated cell sorting (FACS) experiments were performed on a BD FACSAria III flow cytometer. Mock-infected monocytes were used to define the negative gate. For monocytes infected with AF488-DENV2, h3H5-opsonized AF488-DENV2, or polyclonal serum-opsonized AF488-DENV2, a positive gate was used to define the infected cell population, and only a subset of cells within a restricted range of mean fluorescence intensity was sorted for further analysis. A minimum of 5,000 cells were sorted directly into RNeasy RLT lysis buffer (Qiagen) and stored at −80°C.

Chan C.Y., Low J.Z., Gan E.S., Ong E.Z., Zhang S.L., Tan H.C., Chai X., Ghosh S., Ooi E.E, & Chan K.R. (2019). Antibody-Dependent Dengue Virus Entry Modulates Cell Intrinsic Responses for Enhanced Infection. mSphere, 4(5), e00528-19.

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Profiling Rheumatoid Arthritis Synovial Cells

Cited 2 times

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The multicenter RA/SLE (systemic lupus erythematosus) Network of the AMP consortium enrolled individuals meeting the ACR 2010 RA classification according to protocols approved by the IRB at each site (22 (link), 47 (link)). Synovial tissues were collected from ultrasound-guided biopsies or joint replacement surgery and viably frozen in CryoStor CS10 cryopreservation media (Sigma-Aldrich). At a central processing site, tissues were dissociated and cells were FACS (fluorescence-activated cell sorting)–sorted (BD FACSAria Fusion) into fibroblast, macrophage, B cell, and T cell populations. Macrophages were sorted on the basis of CD14⁺CD45⁺ cell surface expression. For bulk RNA-seq on CD14⁺ synovial cell populations, ~1000 cells were sorted directly into RNeasy RLT lysis buffer (Qiagen). For CD14⁺ synovial scRNA-seq, ~100 live cells per patient were individually plated and lysed in 384-well plates.

Kuo D., Ding J., Cohn I.S., Zhang F., Wei K., Rao D.A., Rozo C., Sokhi U.K., Shanaj S., Oliver D.J., Echeverria A.P., DiCarlo E.F., Brenner M.B., Bykerk V.P., Goodman S.M., Raychaudhuri S., Rätsch G., Ivashkiv L.B, & Donlin L.T. (2019). HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness. Science translational medicine, 11(491), eaau8587.

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Quantifying Gene Expression in Ciona Embryos

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Dechorionated Ciona embryos were developed until the appropriate stage. For each sample several hundred embryos were lysed in 330 µL RNeasy RLT lysis buffer (Qiagen, Hilden, Germany). Total RNA was isolated from the lysate using RNeasy mini columns according to the manufacturer’s instructions for animal tissue and eluted in 50 µL RNase free water. cDNA synthesis was performed on 2.5 µg total RNA per sample in a 20 µL reaction using the SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 2.5 µM random hexamers. The reaction conditions were: 23°C for 10 minutes, 50°C for 20 minutes, 80°C for 10 minutes. cDNA samples were then stored at −20°C until processing. qPCR was performed in 25 µl reactions containing 0.5 µl cDNA, 5 pmol of each oligonucleotide primer and 12.5 µL of Power SYBR Green PCR Master Mix (Life Technologies, Warrington, UK). qPCR reactions were performed in a ViiA 7 qPCR instrument (Applied Biosystems, Foster City, CA, USA) in a 96-well plate. The reaction conditions were: 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Expression levels were determined by comparing the cT of each sample with the cT of the corresponding sample at the 1-cell stage normalized to the cT of Gapdh. All primers sets were validated by the presence of a single peak when a melt-curve analysis was performed.

Treen N., Heist T., Wang W, & Levine M. (2018). Depletion of maternal Cyclin B3 contributes to zygotic genome activation in the Ciona embryo. Current biology : CB, 28(7), 1150-1156.e4.

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RNA Extraction from Virus Samples

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A total of 500 µL of RNeasy lysis buffer RLT (Qiagen) and 0.02 M Dithiothreitol (DTT) (ThermoFisher) were added to the treated virus with or without PtCl₄ by pipetting up and down. The lysed virus was transferred to a RNeasy column and extracted using the QIAcube supplemented with DNase I, as previously described by the manufacturer (Qiagen). The purified RNA was eluted in 50 µL of RNase-free water, and 40 units of RNasin Plus RNase Inhibitor (Promega, Madison, WI, USA) was added before its storage at −80 °C.

Raymond P., St-Germain F., Paul S., Chabot D, & Deschênes L. (2024). Impact of Nanoparticle-Based TiO2 Surfaces on Norovirus Capsids and Genome Integrity. Foods, 13(10), 1527.

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RT-qPCR RNA Isolation and Relative Transcript Levels

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Example 11

For the determination of relative transcript levels in RT-qPCR RNA was isolated from 10⁶cultured human ovary adenocarcinoma (SK-OV3) cells, fixed in 1 ml of a fixative composition according to the invention (50% [v/v] Ethanol, 6% [w/v] acetic acid, 20% [v/v] Diethylene glycol monoethyl ether acetate, ad 100% with ddH₂O, pH unadjusted). After 5, 10, 20 or 60 min RNA was isolated from the cells. As a reference for delta Ct value calculation, RNA was also isolated from a cell pellet of 10⁶cells directly lysed in RNeasy lysis buffer RLT (Qiagen) without incubation in the fixative composition.

One step quantitative RT-PCR assays were performed for amplicons within the mRNAs from p53, IL8, cFos, IL1β, Bactin and 18s genes. Ct values are shown as delta Cts. Delta Cts were calculated as Ct values obtain from RNA fixed for 5, 10, 20 or 60 min in the fixative composition according to the invention minus the Ct value obtained with RNA from directly lysed cells.

Delta-Ct values from RT-qPCR are shown in FIG. 11.

US10794803B2. Fixative composition for cell-comprising liquid samples and methods and kit thereof (2020-10-06). QIAGEN GMBH [DE]. Inventors: Daniel Grölz [DE].

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RNA-seq Library Preparation from PBMCs

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PBMCs were lysed with Qiagen RNeasy lysis buffer (RLT) following 24 h of stimulation and stored at −80°C until isolation. RNA was isolated using the Qiagen RNeasy kit, following the manufacturer’s instructions. A total of 200 ng RNA per sample was used for the preparation of RNA sequencing libraries using the KAPA RNA HyperPrep kit with RiboErase (human/mouse/rat [HMR]) (Kapa Biosystems). In short, oligonucleotide hybridization and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94°C for 6 min 30 s. First-strand synthesis, second-strand synthesis, and A-tailing were performed according to protocol. For adapter ligation, a 7-μM stock was used (NEXTflex DNA barcodes; Bioo Scientific). The first and second postligation cleanups were performed according to protocol. For library amplification, 6 cycles were used. Library amplification cleanup was performed using a 0.8× bead-based cleanup. The library size was determined using the high-sensitivity DNA bioanalyzer (Agilent Technologies), the library concentration was measured using the DeNovix double-stranded DNA (dsDNA) high-sensitivity assay. Sequencing was performed using an Illumina NextSeq 500 instrument; 38-bp paired-end reads were generated.

Geckin B., Zoodsma M., Kilic G., Debisarun P.A., Rakshit S., Adiga V., Ahmed A., Parthiban C., Kumar N.C., D’Souza G., Baltissen M.P., Martens J.H., Domínguez-Andrés J., Li Y., Vyakarnam A, & Netea M.G. (2023). Differences in Immune Responses in Individuals of Indian and European Origin: Relevance for the COVID-19 Pandemic. Microbiology Spectrum, 11(2), e00231-23.

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West Nile Virus RNA Quantification

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Pinhead-sized blood cruor was homogenized (2 min at 30 Hz; TissueLyser II, Qiagen, Hilden, Germany) in 600 µL RNeasy Lysis Buffer (RLT) (Qiagen) plus 6 µL ß-mercaptoethanol together with one 5 mm steel bead. Viral RNA was then extracted using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
The organ samples (brain, heart, kidney, liver, spleen, lung, bursa cloacalis, and feather pulp) were also homogenized with one 5 mm steel bead. For RNA extraction, only 100 µL of the homogenized organs and the swab samples were used in the BioSprint 96 (Qiagen) with the NucleoMag VET Kit (Macherey-Nagel, Düren, Germany). All of the RNA extracts were examined with the specific WNV RT-qPCR assay targeting the 5′ untranslated region (5′ UTR) for the simultaneous detection of lineage 1 and 2 strains [34 (link)]. For the quantification of viral RNA copies in each sample, a calibration curve of synthetic WNV RNA was run in parallel using 6-fold serial dilutions [34 (link)]. Additionally, the native virus was diluted, extracted, and used to estimate the TCID₅₀/mL.

Reemtsma H., Holicki C.M., Fast C., Bergmann F., Eiden M., Groschup M.H, & Ziegler U. (2022). Pathogenesis of West Nile Virus Lineage 2 in Domestic Geese after Experimental Infection. Viruses, 14(6), 1319.

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Profiling Splenic Dendritic Cells

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For bulk-RNAseq, splenic DC from 2 mice were CD135-enriched and split in
two samples: 30% was stained to sorting, and 70% was stimulated with the LPRC
adjuvant cocktail [100ng/mL LPS, 25μg/mL Poly(I:C), 2.5μg/mL
Resiquimod and 6μg/mL CpG-A (ODN 2216)] at a concentration of
10⁷/mL for 3 hrs before staining and sorting. RNA was extracted
using the NucleoSpin RNA XS kit (Takara Bio). For scRNAseq, CD135-enriched cells
were stained and sorted. Splenocytes were mixed 1:1 from 2 sorted gates to
enrich for tDC:
CD3⁻CD19⁻NK1.1⁻Ly6G⁻and
CD3⁻CD19⁻NK1.1⁻Ly6G⁻XCR1⁻CD11b^lo(Fig.1c). BM cells were sorted as
CD3⁻CD19⁻NK1.1⁻Ly6G⁻.
600 sorted cells per μL were processed for 10X Genomics. For NanoString,
sorted DC were resuspended in 1/3 RNeasy Lysis Buffer RLT (Qiagen) at a
concentration of 1,000-5,000 cells/μL and analyzed on the NanoString
nCounter^® Mouse Myeloid Innate Immunity V2 Standard
Platforms, following the manufacturer's instructions. Samples were
processed on the NanoString Digital Analyzer to yield a reporter code count
(RCC) dataset, which was analyzed viaROSALIND^® (https://rosalind.bio).

Sulczewski F.B., Maqueda-Alfaro R.A., Alcántara-Hernández M., Perez O.A., Saravanan S., Yun T.J., Seong D., Hornero R.A., Raquer-McKay H.M., Esteva E., Lanzar Z.R., Leylek R.A., Adams N.M., Das A., Rahman A.H., Gottfried-Blackmore A., Reizis B, & Idoyaga J. (2023). TRANSITIONAL DENDRITIC CELLS ARE DISTINCT FROM CONVENTIONAL DC2 PRECURSORS AND MEDIATE PRO-INFLAMMATORY ANTIVIRAL RESPONSES. Nature immunology, 24(8), 1265-1280.

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RNA Isolation and cDNA Synthesis

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Cells were washed twice with PBS and lysed in 350μL RNeasy Lysis Buffer (RLT; Qiagen) supplemented with ß–mercaptoethanol (10μL/mL). After harvesting the lysate, RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. RNA concentrations were measured by UV spectrophotometry using a NanoDrop (Thermo Scientific). RNA was mixed with Quanta qScript cDNA mix (Quanta Biosciences) and water. The reaction was run for 5min at 22°C, 30min at 42°C and 5min at 85°C.

Lang M.B., Leung K.Y., Greene N.D., Malone K.M., Saginc G., Randi A.M., Kiprianos A., Maughan R.T., Pericleous C, & Mason J.C. (2023). The actions of methotrexate on endothelial cells are dependent on the shear stress-induced regulation of one carbon metabolism. Frontiers in Immunology, 14, 1209490.

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RNA-seq Library Preparation from PBMCs

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