Rneasy r mini kit

Manufactured by Qiagen

Sourced in Germany

The RNeasy R Mini Kit is a laboratory equipment designed for the purification of total RNA from various biological samples. It employs a silica-membrane-based technology to efficiently capture and purify RNA molecules, making it suitable for a wide range of applications that require high-quality RNA.

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8 protocols using rneasy r mini kit

Wnt Signaling Pathway Gene Expression in K562 Cells

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Proliferating K562 RepID WT and KO cells were harvested, and RNA was extracted using the RNeasy R Mini Kit (Qiagen, 74104) and converted to cDNA by using PrimeScript RT Master Mix (Takara, RR036A) according to the manufacturer’s instructions. An RT² Profiler PCR array (Qiagen, PAMM-043Z) with 84 genes associated with the Wnt signaling pathway was used.
Total RNA extraction, cDNA synthesis, and qRT-PCR were performed according to the manufacturer’s instructions provided by Qiagen (RNeasy R Mini Kit, 74104), Takara (PrimeScript RT Master Mix RR036A), and Invitrogen (SYBR Green PCR Master Mix 4309155), respectively. The expression levels of human DAB2, CD41, and CD61 in the cells were determined by qRT-PCR, using GAPDH as an internal control. DAB2 primers; forward:5’-CCA GAT GCA AGA GGG GAT AA-3’ and reverse:5’-TCC TCC ACA CAC GTA ACC AA-3,’ CD41 primers; forward:5’-AGG TGA GAG GGA GCA GAA CA-3’ and reverse:5’-TCC ACC TTG AGA GGG TTG AC-3,’ CD61 primers; forward:5’-GAC AAG GGC TCT GGA GAC AG-3’ and reverse:5’-ACT GGT GAG CTT TCG CAT CT-3,’ GAPDH primers; forward:5’-GAG TCA ACG GAT TTG GTC GT-3’ and reverse:5’-TTG ATT TTG GAG GGA TCT CG-3.’

Jo J.H., Ok S.M., Kim D.K., Kim Y.M., Park J.U., Jung D.H., Kim H.J., Seong H.A., Cho H.J., Nah J., Kim S., Fu H., Redon C.E., Aladjem M.I, & Jang S.M. (2023). RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter. Research Square.

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RNA Extraction and Microarray Analysis of Mouse Brain

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Total RNA was extracted from mouse brain using Trizol reagent (Invitrogen), and further purified using RNeasyR Mini Kit (Qiagen) as described previously^{35 (link)}. The quality of RNA was initially assessed by electrophoresis on a 1.5% agarose gel, and further determined by absorption spectrophotometer (Agilent Bioanalyzer 2100 [Agilent, Palo Alto, CA]). cDNAs were synthesized by Low Input Quick Amp Labeling Kit. Cy3-Labeled cRNA was synthesized by in vitro transcription with T7 RNA Polymerase. Following fragmentation, 0.6 μg of cRNA was hybridized for 17 hours at 65 °C on the SurePrint G3 Mouse GE 8 × 60 K Microarray using Gene Expression Hybridization Kit. GeneChips were washed using the Gene Expression Wash Buffers Pack and scanned using Agilent DNA Microarray Scanner (G2565CA). Microarray data was processed using GeneChip Operating Software (Feature Extraction).

Yamasaki T., Deki-Arima N., Kaneko A., Miyamura N., Iwatsuki M., Matsuoka M., Fujimori-Tonou N., Okamoto-Uchida Y., Hirayama J., Marth J.D., Yamanashi Y., Kawasaki H., Yamanaka K., Penninger J.M., Shibata S, & Nishina H. (2017). Age-dependent motor dysfunction due to neuron-specific disruption of stress-activated protein kinase MKK7. Scientific Reports, 7, 7348.

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Microarray Analysis of Mouse Liver Transcriptome

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Mouse livers were harvested at 9 AM–11 AM. Fresh liver samples were immersed in RNALater (Ambion) overnight. Afterwards, the samples were extracted with STAT-60^TM (Tel-Test, Inc.) to retrieve total RNA. The RNA was purified further with RNeasy^R Mini Kit (QIAGEN). An RNA Analyzer (Agilent) was used to check RNA quality. RNAs with high quality were reverse transcribed and the cDNAs were labeled and hybridized to the Affymetrix GeneChip mouse genome 2.0 array. Gene expression data generated by the Affymetrix MOE430 chip (two samples per condition) were imported to GeneSpring for analysis. Absence and presence call for each probe was made by MAS5. To identify differentially expressed genes between any 2 conditions (fold-change >1.5 and p-value < 0.05; t-test), probes labeled by absence on both duplicates in either condition were discarded.

Wang R.H., Zhao T., Cui K., Hu G., Chen Q., Chen W., Wang X.W., Soto-Gutierrez A., Zhao K, & Deng C.X. (2016). Negative reciprocal regulation between Sirt1 and Per2 modulates the circadian clock and aging. Scientific Reports, 6, 28633.

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Quantitative Analysis of Gene Expression

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Total RNA was extracted from hTERT-RPE cells treated with DMSO or BFA using RNeasy^R mini kit (Qiagen). DMSO or BFA was treated 30 min before serum-restimulation at the indicated concentrations. cDNA was generated by SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Conventional RT-PCR was performed using a ProFlex™ Base Thermal Cycler (Applied Bio-systems) with the conditions of 95°C for 20 s, 62°C for 30 s, and 72°C for 45 s for a total of 25 cycles for Plk1, Dvl2, and GAPDH, followed by a 10 min final extension at 72°C. PCR products were electrophoresed in 3% agarose gel, stained with EcoDye™ Nucleic Acid Staining Solution (BIOFACT, Korea), and photographed. Real-time RT-PCR was performed in a final volume of 20 μl with 2 μl of cDNA, 10 pmol forward and 10 pmol reverse primer in 1X power SYBG green PCR master Mix (Applied Biosystems, USA) with the condition of 95°C for 15 s for denaturation, 55°C for 1 min for annealing and 72°C for 15 s extension using an QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). The expression value of each gene was normalized by that of GAPDH. Final values were calculated using the ΔΔCt method. The results were analyzed using QuantStudio™ design & Analysis software v1.4 (Applied Biosystems). All the primers used in these experiments are summarized in Supplementary Table S3.

Lee U., Kim S.O., Hwang J.A., Jang J.H., Son S., Ryoo I.J., Ahn J.S., Kim B.Y, & Lee K.H. (2017). The Fungal Metabolite Brefeldin A Inhibits Dvl2-Plk1-Dependent Primary Cilium Disassembly. Molecules and Cells, 40(6), 401-409.

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Hematopoietic Stem Cell Isolation and Culture

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We isolated BM mononuclear cells by Ficoll-Hypaque gradient centrifugation (Amersham Biosciences) and positively selected BM CD34 + cells by sequential immunomagnetic labeling with anti-CD34 + magnetic cell-sorting beads (Miltenyi Biotech). We assessed cell viability by trypan blue dye exclusion. The purity of the sorted CD34 + cells was more than 95% as determined by FACS analysis.
Colony-forming unit assay CD34 + cells were resuspended in IMDM supplemented with 2% FBS (Stemcell Technologies, #07700) and enriched MethoCult TM (Stemcell Technologies, #H4435). The cell suspension was plated on 3.5 cm dishes (1 × 10 3 cells/dish) for 14 days.
mRNA isolation and cDNA amplification RNA was isolated using the RNeasy R Mini Kit (Qiagen, #74106) according to the manufacturer's instructions. cDNA was amplified using the Ovation R Pico WTA system 2 (NuGEN, San Carlos, CA), which amplifies cDNA out of a small quantity of RNA.

Mir P., Klimiankou M., Findik B., Hähnel K., Mellor-Heineke S., Zeidler C., Skokowa J, & Welte K. (2020). New insights into the pathomechanism of cyclic neutropenia. Annals of the New York Academy of Sciences, 1466(1).

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Wnt Signaling Pathway Gene Expression in K562 Cells

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Jo J.H., Park J.U., Kim Y.M., Ok S.M., Kim D.K., Jung D.H., Kim H.J., Seong H.A., Cho H.J., Nah J., Kim S., Fu H., Redon C.E., Aladjem M.I, & Jang S.M. (2023). RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter. Cell Communication and Signaling : CCS, 21, 219.

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Total RNA Extraction and qPCR Analysis

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Total RNA was column-purified with the RNeasy ○ R Mini Kit (74106; Qiagen) and treated with RNase-free DNase (79254; Qiagen). Purified RNA was reverse transcribed using PrimeScript™ RT Master Mix (RR037A; Takara) according to the manufacturer's protocol. qPCR was performed using StepOnePlus™ (Applied Biosystems) with TB Green Premix Ex Taq II (RR820A; Takara). The primer sequences used in

Kase N., Terashima M., Ohta A., Niwa A., Honda‐Ozaki F., Kawasaki Y., Nakahata T., Kanazawa N., & Saito M.K. (2020). High-throughput Screening with Pluripotent Stem Cells Identifies CUDC-907 as an Effective Compound for Restoring the Proinflammatory Phenotype of Nakajo-Nishimura Syndrome.

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Transcriptome Analysis of Fish Liver

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To evaluate the effects of fish oil on the genes expressed in the liver, the transcriptome analysis was carried out. First, total RNA (n = 6 per group) from the two groups was extracted from fish liver using RNeasy R Mini Kit reagents according to the instructions of the manufacturer (Qiagen GMBH, Hilden, Germany). The RNA quality was determined and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States) and checked using the RNase-free agarose gel electrophoresis. The mRNA was enriched by oligo (dT) beads; the mRNA was interrupted. The first strand of cDNA was synthesized in M-MuLV reverse-transcriptase system, and the second strand was synthesized from dNTPs in DNA polymerase I system. Then, the cDNA fragments were ligated to Illumina sequencing adapters. The ligation products were size selected using agarose gel electrophoresis, PCR amplified, and sequenced using novaseq 6000 by Guangzhou Genedenovo Biotechnology Co. (Guangzhou, China). All clean libraries of sequencing data were submitted to the NCBI Sequence Read Archive (SRA) database (Accession No: PRJNA765892).

Wang T., Jiang D., Shi H., Mustapha U.F., Deng S., Liu Z., Li W., Chen H., Zhu C., & Li G. (2021). Liver Transcriptomic Analysis of the Effects of Dietary Fish Oil Revealed a Regulated Expression Pattern of Genes in Adult Female Spotted Scat (Scatophagus argus). Frontiers in Marine Science, 8.

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