Rneasy powerlyzer tissue cells kit

Manufactured by Qiagen

Sourced in United States

The RNeasy PowerLyzer Tissue & Cells Kit is a product designed for the isolation and purification of total RNA from a variety of tissue and cell samples. It utilizes a mechanical lysis method to efficiently disrupt samples and release the RNA, followed by a silica-membrane-based purification process to capture and purify the RNA.

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Lab products found in correlation

Hiseq 2500, by Illumina (2 mentions) Bioanalyzer 2100 system, by Agilent Technologies (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Dneasy purification kit, by Qiagen (1 mentions) Onestep pcr inhibitor removal kit, by Zymo Research (1 mentions) Direct zol rna miniprep w zymo spin iic columns, by Zymo Research (1 mentions) Nebnext rrna depletion kit, by New England Biolabs (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Agilent bioanalyzer, by Agilent Technologies (1 mentions) Direct zol rna miniprep plus, by Zymo Research (1 mentions) Kapa rna hyperprep kit with riboerase, by Roche (1 mentions) Truseq rna sample prep kit, by Illumina (1 mentions) Rnaqueous 4pcr total rna isolation kit, by Thermo Fisher Scientific (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Truseq rna exome kit, by Illumina (1 mentions) Qubit rna broad range assay, by Thermo Fisher Scientific (1 mentions) Tape station bioanalyzer, by Agilent Technologies (1 mentions) Nebnext ultra 2 rna library prep kit for, by Illumina (1 mentions) Novaseq platform, by Illumina (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)

8 protocols using rneasy powerlyzer tissue cells kit

Melanoma Sequencing and RNA Extraction

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Melanomas were sequenced from snap-frozen tumors (Yale and CSU cohorts) or low passage cell cultures (<4) as previously described^{2 (link),4 (link)} (Supplementary Data 1, 8). DNA from melanoma cells and freshly frozen tumors was extracted with the DNeasy purification kit (Qiagen Inc., Valencia, CA). High melanin content was removed with OneStep™ PCR Inhibitor Removal Kit (Zymo Research Corporation, Irvine, CA). Direct-zol™ RNA MiniPrep w/ Zymo-Spin™ IIC Columns (Zymo catalog no. D4019) were used to extract RNA from tumors, and the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen, catalog no. 15055-50) was used to extract RNA from peripheral blood mononuclear cells (PBMCs) and melanoma cells.

Farshidfar F., Rhrissorrakrai K., Levovitz C., Peng C., Knight J., Bacchiocchi A., Su J., Yin M., Sznol M., Ariyan S., Clune J., Olino K., Parida L., Nikolaus J., Zhang M., Zhao S., Wang Y., Huang G., Wan M., Li X., Cao J., Yan Q., Chen X., Newman A.M, & Halaban R. (2022). Integrative molecular and clinical profiling of acral melanoma links focal amplification of 22q11.21 to metastasis. Nature Communications, 13, 898.

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Stigma Transcriptome Profiling Protocol

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Deep-frozen stigma samples were retrieved from −80°C and 200 µL of 1× PBS (pH 7.4, Invitrogen, Carlsbad, CA) + 0.001% Silvet L77 (PlantMedia, Dublin, OH) + beta-mercaptoethanol (% vol/vol) were added to the tube. Epiphytic bacteria were detached by combining water bath sonication for 5 min and followed by full-speed vortex for 30 s as described in the DNA extraction protocol. Stigma was removed from the tube, and RNA was extracted using the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Yield and quality were determined using QuBit and Agilent Bioanalyzer, respectively. Eluted RNA was depleted from ribosomal RNA using NEBNext rRNA Depletion Kit (Bacteria, New England Biolabs, MA), then used as a template to generate cDNA using NEBNext Ultra RNA Library Prep Kit for Illumina (England Biolabs, Ipwich, MA). The resulting cDNA was assessed with a high-sensitivity Agilent 2100 bioanalyzer. Size profiles of cDNA fragments were consistent across samples.

Hassani M.A., Cui Z., LaReau J., Huntley R.B., Steven B, & Zeng Q. (2024). Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity. mBio, 15(3), e00213-24.

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RNA Isolation from Tissue and Cells

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RNA from spleen, aortic arch, and cultured cells was isolated using the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen) and Direct-zol™ RNA Miniprep Plus (Zymo Research) for tissue and cells, respectively, according to the manufacturer’s protocol. A full description of RT-PCR is found in the Supplementary Material section.

Ray M., Gabunia K., Vrakas C.N., Herman A.B., Kako F., Kelemen S.E., Grisanti L.A, & Autieri M.V. (2018). Genetic Deletion of IL-19 Exacerbates Atherogenesis in Il19−/− × Ldlr−/− Double Knockout Mice by Dysregulation of mRNA Stability Protein HuR. Arteriosclerosis, thrombosis, and vascular biology, 38(6), 1297-1308.

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Bulk RNA Sequencing of Melanoma Tumors

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Bulk RNA sequencing was performed on melanoma tumors, as previously described^{37 (link),38 (link)}. In brief, RNA was extracted from tumors using the RNeasy PowerLyzer Tissue & Cells Kit (QIAGEN) and quality was assessed with a 2100 Bioanalyzer System (Agilent Technologies). Ribosomal RNA was depleted Kapa RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Inc., Cape Town, South Africa). Sequencing libraries were generated using TruSeq RNA sample prep kits (Illumina) and sequenced on an Illumina HiSeq 2500 using 2 × 150 bp paired-end reads with a target of 20–25 million reads per sample. Raw sequencing files were aligned to the GRCh38 human genome.

Hafler D., Lu B., Lucca L., Lewis W., Wang J., Nogeuira C., Heer S., Axisa P.P., Buitrago-Pocasangre N., Pham G., Kojima M., Wei W., Aizenbud L., Bacchiocchi A., Zhang L., Walewski J., Chiang V., Olino K., Clune J., Halaban R., Kluger Y., Coyle A., Kisielow J., Obermair F.J, & Kluger H. (2024). Circulating Tumor Reactive KIR+CD8+ T cells Suppress Anti-Tumor Immunity in Patients with Melanoma. Research Square.

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Quantifying Gene Expression in Mangosteen-Treated Cancer Cells

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22Rν1 cells (1 × 10⁶) were plated in 6-well plates and incubated for 24 h. α-Mangostin was added to the plates at either 10 or 20 μM concentrations for 24 h. RNA was then extracted from the cells using an RNAqueous™-4PCR Total RNA Isolation Kit (#AM1914, Invitrogen, Waltham, MA, USA) by following the recommended protocol. RNA was resuspended in nuclease-free water.
RNA was extracted from tumor tissues using an RNeasy PowerLyzer Tissue & Cells kit (#15055, Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions, and RNA was resuspended in nuclease-free water.
cDNA was made with the iScript™ cDNA Synthesis kit (#1708890, Bio-Rad), according to the manufacturer’s instructions. qPCR was run using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. All primers were designed using the NCBI-Primer Blast tool and ordered from Integrated DNA Technologies (Coralville, IA, USA).

Nauman M.C., Won J.H., Petiwala S.M., Vemu B., Lee H., Sverdlov M, & Johnson J.J. (2023). α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells. Cancers, 15(7), 2118.

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Transcriptomic Analysis of Cryopreserved Cells

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Cryopreserved cell suspensions were thawed as described above. RNA was subsequently extracted using the RNeasy PowerLyzer Tissue & Cells Kit (QIAGEN) and quality was assessed with a 2100 Bioanalyzer System (Agilent Technologies). All samples were sufficiently high quality for TruSeq RNA Exome analysis (DV₂₀₀ > 30%) and were prepared using the TruSeq RNA Exome Kit (Illumina) according to the manufacturer’s instructions. After hybrid capture, cDNA libraries were pooled and sequenced on a HiSeq 2500 instrument (Illumina) using 2 × 150 bp paired-end reads with a target of 20–25 million reads per sample. Raw reads were quantified with Salmon v.0.12.0 using the GENCODE v.29 reference transcriptome; the following command line arguments were used with otherwise default parameters: --seqBias--gcBias--posBias--validateMappings--rangeFactorizationBins 4. Read counts were normalized to gene-level transcripts per million (TPM) using tximport v.1.10.1 (ref.^{73 (link)}). Only samples with a mapping rate ≥60% and successful TCR assembly (see the V(D)J receptor profiling and clonotype analysis below) were included for further analysis, with the exception of 3 samples with mapping rates >40% (but < 60%) and successful TCR assembly, which were included. In total, 53 sequenced samples (88%) in bulk cohorts 1 and 2 satisfied these criteria (Fig. 1 and Supplementary Table 8).

Lozano A.X., Chaudhuri A.A., Nene A., Bacchiocchi A., Earland N., Vesely M.D., Usmani A., Turner B.E., Steen C.B., Luca B.A., Badri T., Gulati G.S., Vahid M.R., Khameneh F., Harris P.K., Chen D.Y., Dhodapkar K., Sznol M., Halaban R, & Newman A.M. (2022). T cell characteristics associated with toxicity to immune checkpoint blockade in patients with melanoma. Nature medicine, 28(2), 353-362.

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Bacterial Transcriptomics: rRNA Depletion and Illumina Sequencing

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Bacterial cells were collected from mono- and co-cultures after 6 h of growth at 28°C in a partial stigma-mimicking medium. RNA was extracted using the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen) according to the manufacturer’s instructions. The RNA was eluted in 50 µL nuclease-free water. Quantity and quality control of the RNA was assessed by using Qubit RNA Broad-Range Assay (Invitrogen) and with RNA ScreenTape analysis on Agilent TapeStation Bioanalyzer. Before Illumina library preparation, rRNA depletion was conducted using the NEBNext rRNA Depletion Kit (Bacteria, New England Biolabs) according to the manufacturer’s recommended protocol. Then, DNA removal coupled with cDNA synthesis was conducted using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s recommended protocol. DNA quantity was checked using the Qubit dsDNA HS Assay Kit (Invitrogen). Library sequencing was conducted on an Illumina NovaSeq platform through services provided by the Yale Center for Genome Analysis.

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Ovarian HE4 Gene Expression Analysis

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Ovarian tissue samples were obtained from patients by biopsy before and after the intervention. The total RNA was extracted by RNeasy PowerLyzer Tissue & Cells Kit (Qiagen, USA) according to its protocol. After extraction, the quality of RNAs was evaluated by spectrophotometric study of light absorption at 260/280nm. First, 10μl of RNA template was mixed with 1μl of 10Mm dNTP and 1μl of Random Hexamer followed by 1μl of Oligo dt to synthesize cDNA from RNA. The tube was then placed at 65°C for 5 minutes. Then two microliters of MMuLV 10X buffer and 0.5 microliters of M-MuLV enzyme were added to the mixture. Finally, double distilled water was added, and the final volume reached 20μl. The tube was incubated for 1 hour at 42°C. The GAPDH gene sequence (as an internal control) and the HE4 gene sequence were obtained from the NCBI gene bank, and the Express Primer software designed their specific primers. The obtained sequences were blasted at NCBI and Gene Runner to confirm the specificity and accuracy of the designed primers. The primer sequences are listed in Table 1.
Table1. The sequences and the production size of HE4 and GADPH primers

Li G, & Zhang X. (2022). The effect of supportive and educational nursing care on quality of life and HE4 gene expression in patients with ovarian cancer. Cellular and molecular biology (Noisy-le-Grand, France), 68(5).

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