Ez1 rna universal tissue kit

Manufactured by Qiagen

Sourced in Germany

The EZ1 RNA Universal Tissue Kit is a lab equipment product designed for the automated extraction and purification of total RNA from various types of tissue samples. It provides a standardized and streamlined process for RNA isolation, enabling efficient and consistent results.

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8 protocols using ez1 rna universal tissue kit

Transcriptome Profiling of Frozen Tissues

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Total RNA was extracted from frozen tissues after thawing and homogenizing by IKA Ultra-Turrax and QIAzol Lysis Reagent. RNA was extracted with the phenol chloroform method. From the aqueous phase, RNA was automatically purified by BioRobot EZ1using EZ1 RNA Universal Tissue kit instrument according to the manufacturer’s instructions (Qiagen S.p.A., Milan, Italy). RNA was eluted in 50 μl of RNase-free water and stored at − 80 °C until use. The total RNA extracted from the samples was prepped according to the Illumina TruSeq RNA Sample prep kit. Thus, each sample was indexed, pooled by 10, and sequenced on a single lane of Illumina Flow Cell PE v3 (Illumina Inc., CA, USA). Sequencing was performed on Illumina HiSeq1500 with TruSeq SBS chemistry (200 cycles), generating 2 × 100 Paired-Ends reads.

Napolioni V., Bianconi F., Potenza R., Carpi F.M., Ludovini V., Picciolini M., Tofanetti F.R., Bufalari A., Pallotti S., Poggi C., Anile M., Daddi N., Venuta F., Puma F, & Vannucci J. (2021). Genome-wide expression of the residual lung reacting to experimental Pneumonectomy. BMC Genomics, 22, 881.

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Thymus RNA Extraction Protocol

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Thymus from each pup was collected and snap-frozen in liquid N₂ with 5 min after dissection. The samples were kept in a −80 °C freezer until processing for total RNA extraction. Thymus was disrupted using the TissueLyser (Qiagen, Valencia, CA) in the QIAzol Lysis Reagent (Qiagen) and total RNA was isolated on the EZ1 Advanced XL (Qiagen) automated RNA purification instrument using the EZ1 RNA Universal Tissue Kit (Qiagen) following the manufacturer's protocol, including an on-column DNase digestion. RNA concentration and purity (260/280 ratio) were measured with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Integrity of RNA samples was assessed by the Agilent 2100 Bioanalyzer (Santa Clara, CA) with the RNA 6000 Nano Reagent Kit from the same manufacturer.

Gao X., Yourick J.J., Topping V.D., Black T., Olejnik N., Keltner Z, & Sprando R.L. (2014). Toxicogenomic study in rat thymus of F1 generation offspring following maternal exposure to silver ion. Toxicology Reports, 2, 341-350.

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Cardiac and Skeletal Muscle RNA Extraction

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Approximately 50 mg of heart muscle (ventricle) or soleus muscle were homogenized in QIAZOL (Qiagen, Germany) plus 8 µl proteinase K (Qiagen) in a TissueLyser bead mixer (Qiagen) at 25 Hz in two 5-min repetitions. Total RNA isolation was performed with an EZ1 RNA Universal Tissue Kit and Biorobot EZ1 (Qiagen) in accordance with the manufacturer's instructions. Total RNA concentrations were measured at 260 nm via spectrophotometry (ND-1000 spectrophotometer, NanoDrop Technologies, Inc.). Samples were frozen and stored at −80°C for subsequent analysis.

Gilbert A., Wyczalkowska-Tomasik A., Zendzian-Piotrowska M, & Czarkowska-Paczek B. (2016). Training differentially regulates elastin level and proteolysis in skeletal and heart muscles and aorta in healthy rats. Biology Open, 5(5), 556-562.

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Quantitative PCR Analysis of Chylomicron Metabolism

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Euthanized larvae were homogenized on Quiazol (Qiagen) using a Precellys 24 (Bertin Technologies). Total RNA was extracted from the whole fish on a Bio Robot EZ1 using the EZ1 RNA Universal Tissue Kit with the RNase-free DNase Set (Qiagen), according to the manufacturer's instructions. The quantification and purity of RNA was assessed with the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies). For all total RNA samples, the optical density ratio at 260/280 nm ranged between 1.70 and 1.98. RNA integrity numbers [22 (link),23 (link)] were between 8.2 and 9.7.
RT reactions and qPCR were run according to published methods [24 (link)]. Genes coding for proteins involved in assembly and transport of chylomicrons, ER-stress, cytosolic lipid droplet metabolism and mitochondrial ß-oxidation were analyzed (Table 1). Results were calculated as the arithmetic mean using ubiquitin and beta-actin as reference genes based on the study of Sæle et al [24 (link)]. Normalization was performed using the geNorm Visual Basic for Applications applet for Microsoft as previously described by Vandesompele et al [25 (link)]. Accession number, primer sequences and product size for each quantitative PCR assay are given together with the corresponding biological pathway in Table 1.

Sæle Ø., Rød K.E., Quinlivan V.H., Li S, & Farber S.A. (2018). A novel system to quantify intestinal lipid digestion and transport. Biochimica et biophysica acta, 1863(9), 948-957.

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Paraquat-Induced Stress Response in S2 Cells

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S2 cells were subjected to RNAi alone or RNAi and 24 h treatment with 8.3 mM paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride (Sigma Aldrich)). Total RNA was isolated using the EZ1^® RNA Universal Tissue Kit (Qiagen). Cells were disrupted and homogenized in 750 µl QIAzol™ lysis reagent via bead-milling on the TissueLyser^® II (Qiagen). RNA was collected from the homogenate by chloroform extraction and purified on the EZ1^® Advanced (Qiagen) with additional DNase step to remove any residual DNA. Purified total RNA was quantified by UV spectrophotometry using the DropSense96^® Microplate Spectrophotometer (Trinean) and purity was assessed based on the A260/A280 and A260/A230 ratios. RNA quality was assessed by microfluidics using the RNA R6K assay for the Agilent 2200 TapeStation. The electrophoretogram was examined to determine overall quality of the RNA.

Gajan A., Barnes V.L., Liu M., Saha N, & Pile L.A. (2016). The histone demethylase dKDM5/LID interacts with the SIN3 histone deacetylase complex and shares functional similarities with SIN3. Epigenetics & Chromatin, 9, 4.

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RNA Extraction and Sequencing of Naegleria fowleri

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RNA was extracted from 10⁷
N. fowleri trophozoites cultivated in Nelson’s medium using the EZ1 RNA Universal Tissue Kit (Qiagen) and the EZ1 BioRobot (Qiagen). Trophozoites were resuspended in 750 μl of QIAzol lysis reagent (Qiagen), followed by disruption and homogenization by operating a TissueLyser at 25 Hz for 3 min. After incubation for 5 min at room temperature, 150 μl chloroform (Grogg, Stettlen, Switzerland) was added to the homogenized samples. The mixture was then centrifuged for 15 min at 12,000 g at 4°C, and the upper aqueous phase was used as the starting material for RNA isolation with the EZ1 BioRobot, according to manufacturer’s protocol. Quantification and examination of the total RNA integrity was performed with the Agilent 2100 Bioanalyzer system. Four micrograms of high-quality RNA was sent to the Next Generation Sequencing Platform of the University of Bern for paired-end sequencing by the Illumina HiSeq 2000 device.
The reads from RNA sequencing have been deposited in DDBJ/EMBL/GenBank under accession SRX553040.

Zysset-Burri D.C., Müller N., Beuret C., Heller M., Schürch N., Gottstein B, & Wittwer M. (2014). Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri. BMC Genomics, 15(1), 496.

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RNA Isolation from N. lovaniensis

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For total RNA isolation, N. lovaniensis was cultivated in the media Nelson, PYNFH and PYNFH supplemented with Liver Hydrolysate. Trophozoites were harvested in the late log phase by centrifugation and washed three times using PBS. For cell disruption, trophozoites were resuspended in 750 μl QIAzol lysis reagent (Qiagen) and homogenized in the TissueLyser (Qiagen) for 3 min at 25 Hz. After incubation at room temperature for 5 min, 150 μl chloroform (Sigma) was added to the sample followed by centrifugation for 15 min at 12,000 g at 4 °C. RNA from the upper aqueous phase was extracted using the EZ1 RNA Universal Tissue Kit (Qiagen) and the EZ1 BioRobot (Qiagen) according to the manufacturer’s protocol. The purified RNA was quantified using the Qubit2.0 Fluorometer with the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and the total RNA integrity was examined using the Agilent 2100 Bioanalyzer system (Agilent Technologies). Total RNA (2 μg) was sent to the NGS Platform at the University of Bern (Bern, Switzerland) for Illumina HiSeq 3000 150 bp paired-end sequencing.

Liechti N., Schürch N., Bruggmann R, & Wittwer M. (2018). The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri. BMC Genomics, 19, 654.

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Lung Gene Expression Analysis

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Gene expression in lung tissue of podoplanin, α‐SMA, fibronectin, and collagen was determined by RT‐PCR as follows. Total RNA was extracted from snap‐frozen lung hemilobes using EZ1 RNA Universal Tissue Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. cDNA was prepared with ImProm‐IITM Reverse Transcription System (Promega, Madison, Wisconsin). Quantitative RT‐PCR was performed using PowerUp Green Master Mix SYBR Green PCR master kit (Life Technologies) on a ABIPRISM 7000 instrument (Applied Biosystems). Data were normalized to the housekeeping gene (β‐actin) and relative gene expression was quantified using the 2^−ΔCT method. Primer sequences are shown in Supporting Information Table S1.

Cargnoni A., Romele P., Bonassi Signoroni P., Farigu S., Magatti M., Vertua E., Toschi I., Cesari V., Silini A.R., Stefani F.R, & Parolini O. (2020). Amniotic MSCs reduce pulmonary fibrosis by hampering lung B‐cell recruitment, retention, and maturation. Stem Cells Translational Medicine, 9(9), 1023-1035.

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