Rneasy midi kit

Manufactured by Qiagen

Sourced in Germany, United States, Spain, France, United Kingdom, Netherlands, Japan

The RNeasy Midi Kit is a laboratory tool designed for the purification of total RNA from a variety of sample types, including cell lines and tissue samples. The kit utilizes a silica-based membrane technology to efficiently capture and purify RNA molecules, enabling users to obtain high-quality RNA for downstream applications.

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RNeasy Mini/Midi Kit RNeasy Plus Universal Midi Kit RNeasy Midi RNA Extraction kit RNeasy Lipid Tissue Midi Kit RNeasy Midi Cleanup Kit RNeasy maxi (lung) or midi (liver) kit RNeasy Fibrous Tissue Midi Kit RNeasy® Mini or Midi kit RNeasy Universal Midi kit RNeasy Midi extraction kit

398 protocols using rneasy midi kit

RNA Isolation from RPTEC/TERT1 Cells

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Total RNA was isolated from RPTEC/TERT1 cells using TRIzol/Chloroform method and by RNEasy Midi Kit (Qiagen, Hilden, Germany). A detailed protocol for RNA isolation by TRIzol method is provided in Additional file 1: Supplementary Methods. For samples prepared using RNEasy Midi Kit (Qiagen), the manufacturer’s recommended protocol was followed. The quality and integrity of all RNA samples were measured by Bioanalyzer-2100 using RNA 6000 Pico kit (Agilent Technologies) according to the manufacturer’s recommended protocol.

Islam M.N., Griffin T.P., Sander E., Rocks S., Qazi J., Cabral J., McCaul J., McMorrow T, & Griffin M.D. (2019). Human mesenchymal stromal cells broadly modulate high glucose-induced inflammatory responses of renal proximal tubular cell monolayers. Stem Cell Research & Therapy, 10, 329.

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Transcriptional Analysis of fab and yycFG Genes in S. pneumoniae

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RNA was prepared from 10 mL cultures following the QIAGEN RNeasy Midi Kit (QIAGEN) procedure. The total RNA concentrations were determined by UV spectrophotometry. The RNAs were checked for integrity and yield by analysis in 1% agarose gel. In addition, the quality and quantity of the RNA samples were checked using an RNA 6000 Nano assay with the Agilent 2100 Bioanalyzer (Agilent Technologies) according to the protocol of the manufacturer.
For transcriptional analysis fab and yycFG gene-specific probes for S. pneumoniae TIGR4 strain were used, made as previously described (Mohedano et al., 2005 (link)).
Fluorescent labeling of RNA and hybridization were performed as previously described (Mohedano et al., 2005 (link)). Analysis was conducted on RNA purified from three independent cultures of each strain. The standard deviation of the mean value of the fold increase or decrease obtained in each hybridization for each induction condition (compared to the TIGR4[pLS1RGFP] control strain grown in the same condition) was calculated and these data are shown in Figure 2A.

Mohedano M.L., Amblar M., de la Fuente A., Wells J.M, & López P. (2016). The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae. Frontiers in Microbiology, 7, 1326.

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Spleen RNA Extraction and cDNA Synthesis

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Tissue samples were taken from the spleen and stored at -80°C until RNA extraction. 100 mg of spleen samples were disrupted and homogenized in RTL buffer (QIAGEN GmbH, Germany) using Ultra-Turrax homogenizer (IKA-Werke GmbH & Co. KG, Germany). Total RNA was extracted using Qiagen RNeasy midi kit (QIAGEN GmbH, Germany), according to the manufacturer’s recommendations and extracted RNA was further treated with a ribonuclease inhibitor (RNasin Plus RNase Inhibitor; Promega Corp., USA). A Nanodrop ND-1000 spectrophotometer (Thermo Fischer Scientific, USA) was used to deteremine the quantity and quality of extracted total RNA. The integrity of RNA was verified by agarose gel electrophoresis. The total RNA isolated from each sample was further used to generate cDNA using M-MuLV Reverse Trascriptase kit (Fermentas, Thermo Fischer Scientific, USA) according to the manufacturer’s protocol [19 (link)].

Pistol G.C., Braicu C., Motiu M., Gras M.A., Marin D.E., Stancu M., Calin L., Israel-Roming F., Berindan-Neagoe I, & Taranu I. (2015). Zearalenone Mycotoxin Affects Immune Mediators, MAPK Signalling Molecules, Nuclear Receptors and Genome-Wide Gene Expression in Pig Spleen. PLoS ONE, 10(5), e0127503.

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RNA Isolation and Integrity Verification

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The total RNA from untreated and LB treated IPEC-cells was isolated using a QiagenRNeasy midi kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s recommendations as described by [52 (link)] and their quality and integrity was verified by using an Agilent 2100 bioanalyzer and Agilent RNA 6000 nano kit (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number (RIN) score ranged between 8 and 10. The purified RNA samples were preserved at −80 °C until used.

Taranu I., Marin D.E., Braicu C., Pistol G.C., Sorescu I., Pruteanu L.L., Berindan Neagoe I, & Vodnar D.C. (2018). In Vitro Transcriptome Response to a Mixture of Lactobacilli Strains in Intestinal Porcine Epithelial Cell Line. International Journal of Molecular Sciences, 19(7), 1923.

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RNA Isolation and Northern Blot Analysis

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Total cellular RNA was isolated from 1x10⁹ G418^S/neo⁺ cells using the Qiagen RNeasy Midi Kit (Qiagen) according to the manufacturer’s protocol. All solutions were treated with Diethylpyrocarbonate (DEPC) or prepared in DEPC treated water. For RNA blot analysis 20ug of total RNA from G418^S/neo⁺ clones were resuspended in 2X loading buffer (50% formamide, 6% Formaldehyde, 1X MOPS, 10% glycerol, 0.05% bromphenol blue) and heated at 65°C for 10 min and cooled. Samples were electrophoresed on a 1% agarose-formaldehyde gel in 1X MOPS buffer for 8 hours under 50 volts. Total RNA was transferred to a positively charged nylon membrane (Roche Biochemical) by capillary transfer in DEPC-treated 20X SSC for 24 hrs. Hybridizations were performed in DIG Easy Hybridization solution with designated DIG-labeled probes. Membranes were analyzed using the DIG Detection system (Roche Biochemicals) and analyzed using the ChemiDoc XRS Photodocumentation System (BioRAD).

Cherry K.E., Hearn W.E., Seshie O.Y., Singleton T.L, & Singleton T.L. (2014). Identification of Tf1 integration events in S. pombe under nonselective conditions. Gene, 542(2), 221-231.

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RNA Extraction from Frozen Spleen

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Frozen spleen tissue samples (100 mg) were disrupted and homogenized in RTL buffer (QIAGEN GmbH, Germany) using Ultra-Turrax homogenizer (IKA-Werke GmbH & Co. KG, Germany). Total RNA was extracted using Qiagen RNeasy midi kit (QIAGEN GmbH, Germany), according to the manufacturer’s recommendations. After extraction, RNA was treated with a ribonuclease inhibitor (RNasin Plus RNase Inhibitor; Promega Corp., USA) and the quantity and quality of extracted total RNA were measured on a Nanodrop ND-1000 spectrophotometer (Thermo Fischer Scientific, USA). The integrity of RNA was verified by agarose gel electrophoresis.

Taranu I., Gras M., Pistol G.C., Motiu M., Marin D.E., Lefter N., Ropota M, & Habeanu M. (2014). ω-3 PUFA Rich Camelina Oil By-Products Improve the Systemic Metabolism and Spleen Cell Functions in Fattening Pigs. PLoS ONE, 9(10), e110186.

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Comprehensive Multi-Omics Analysis of Kidney Samples

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Genomic DNA was extracted using a QiAamp DNA Mini kit (Qiagen, Hilden, Germany, RRID:SCR_008539) from 45 WTPDX, 39 available corresponding primary tumors and three normal adult kidney samples and was used for STR analysis, TERT promoter sequencing, and TERT promoter methylation analysis. In addition, total RNA was extracted from 37 paired primary tumors and WTPDX, eight additional WTPDX, and three normal kidney specimens by using the Qiagen RNeasy Midi kit (Qiagen). Commercially available pooled total RNA from four human fetal kidney specimens was also included (Takara, Kusatsu, Japan) and used for RNA-seq and quantitative real time PCR (qRT-PCR). This specific method for RNA extraction is also stated in a previous publication [20 (link)].

Jablonowski C.M., Gil H.J., Pinto E.M., Pichavaram P., Fleming A.M., Clay M.R., Hu D., Morton C.L., Pruett-Miller S.M., Hansen B.S., Chen X., Jones K.M., Liu Y., Ma X., Yang J., Davidoff A.M., Zambetti G.P, & Murphy A.J. (2022). TERT Expression in Wilms Tumor Is Regulated by Promoter Mutation or Hypermethylation, WT1, and N-MYC. Cancers, 14(7), 1655.

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Total RNA Extraction from Cells

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Total RNA was extracted from HepG2 cells or primary Dravet fibroblasts using the QIAGEN RNeasy Midi Kit (QIAGEN, USA) as described by the manufacturer. PolyA RNA was isolated with the Poly(A)Purist™ MAG Kit and eluted twice with 200 μl of THE RNA Storage Solution (Ambion, USA).

Hsiao J., Yuan T.Y., Tsai M.S., Lu C.Y., Lin Y.C., Lee M.L., Lin S.W., Chang F.C., Liu Pimentel H., Olive C., Coito C., Shen G., Young M., Thorne T., Lawrence M., Magistri M., Faghihi M.A., Khorkova O, & Wahlestedt C. (2016). Upregulation of Haploinsufficient Gene Expression in the Brain by Targeting a Long Non-coding RNA Improves Seizure Phenotype in a Model of Dravet Syndrome. EBioMedicine, 9, 257-277.

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RNA Extraction from Fresh-Frozen and FFPE Samples

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Total RNA was purified from fresh-frozen samples for microarray using the Qiagen RNeasy Midi Kit according to the manufacturer's protocol (Qia-gen, Valencia, CA). The integrity of the RNA was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The High Pure RNA Paraffin Kit (Roche Applied Science, Indianapolis, IN) was used to extract RNA from FFPE tissues (2 ϫ 10-m sections or 1.5-mm punches) for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Contaminating DNA was removed using Turbo DNase (Ambion, Austin, TX). The yield of total RNA was assessed using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc, Rockland, DE).

Parker J.S., Mullins M., Cheang M.C.U., Leung S., Voduc D., Vickery T., Davies S., Fauron C., He X., Hu Z., Quackenbush J.F., Stijleman I.J., Palazzo J., Marron J.S., Nobel A.B., Mardis E., Nielsen T.O., Ellis M.J., Perou C.M, & Bernard P.S. (2023). Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 41(26).

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Microglia RNA Extraction from Brain Regions

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Microglia purified from individual brain regions were immediately processed for RNA extraction using the RNeasy Plus Micro Kit (Qiagen, UK). Preliminary experiments showed this method produced the highest yield and quality of RNA. RNA was extracted according to the manufacturer’s instructions with the exception of the final step where RNA elution was repeated twice with 10 μl RNase-free water. RNA quantities were determined by Nanodrop 1000 (Thermo Fisher Scientific, MA, USA) and RNA quality assessed using the Agilent Bioanalyzer (Agilent Technologies, CA, USA). RNA was also extracted from regional mixed brain cell homogenates using the RNeasy Midi Kit (Qiagen) following manufacturer’s instructions. All samples passed a quality control threshold (RIN ≥ 8) to proceed to microarray.

Grabert K., Michoel T., Karavolos M.H., Clohisey S., Baillie J.K., Stevens M.P., Freeman T.C., Summers K.M, & McColl B.W. (2016). Microglial brain region-dependent diversity and selective regional sensitivities to ageing. Nature neuroscience, 19(3), 504-516.

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