Dynabeads mrna direct purification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Netherlands

The Dynabeads mRNA DIRECT Purification Kit is a laboratory product designed for the isolation and purification of messenger RNA (mRNA) from biological samples. The kit utilizes magnetic beads coated with oligo(dT) to capture and isolate polyadenylated mRNA molecules.

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Spelling variants of this product

Dynabeads (2112 citations) MRNA purification kit (13 citations) MRNA Direct kit (9 citations) Dynabeads mRNA DIRECT (5 citations) MRNA direct purification kit (2 citations) Dynabeads mRNA kit (2 citations) Dynabead mRNA DIRECT Micro Purification Kit (2 citations)

154 protocols using dynabeads mrna direct purification kit

qPCR Analysis of Immune Genes in DCs

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RNA was isolated from 5,000 to 10,000 DC previously facs-sorted into 40 μl of lysis buffer (Life Technologies). Dynabeads mRNA Direct Purification Kit (Life Technologies) was used following manufacturer's guidelines. RNA was reverse transcribed with High Capacity cDNA Transcription Kit (Applied Biosystems). PCR were performed with Platinum SYBR Green qPCR SuperMix (Life Technologies) and QuantStudio 6 Flex (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the cycle threshold values (Ct), in duplicate, of the gene of interest with the Ct values of the TBP housekeeping gene.
Primers used in the research: Il6 Fwd 5′-CCTCTCTGCAAGAGACTTCCAT-3′, Il6 Rev 5′-ACAGGTCTGTTGGGAGTGGT-3′, Il12a (p35) Fwd 5'-GCCACCCTTGCCCTCCTAA-3', Il12a (p35) Rev 5'-GGTTTGGTCCCGTGTGATGTC-3', Irf1 Fwd 5′-GTTGTGCCATGAACTCCCTG-3′, Irf1 Rev 5'-TGGACTTTCTCTCTTTCCTCTGG-3′, Ccr7 Fwd 5′-CTCCTTGTCATTTTCCAGGTGTG-3′, Ccr7 Rev 5′-GGCAGGAACCAGGCCTTAAA-3′, Tbp 5′-GAAGCTGCGGTACAATTCCAG-3′, Tbp Rev 5'-CCCCTTGTACCCTTCACCAAT-3′.

Curato C., Bernshtein B., Zupancič E., Dufner A., Jaitin D., Giladi A., David E., Chappell-Maor L., Leshkowitz D., Knobeloch K.P., Amit I., Florindo H.F, & Jung S. (2019). DC Respond to Cognate T Cell Interaction in the Antigen-Challenged Lymph Node. Frontiers in Immunology, 10, 863.

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Comprehensive TCR Sequencing Protocol

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mRNA was isolated using the Dynabeads mRNA DIRECT Purification Kit (Life Technologies) and was amplified using the MessageAmp II aRNA Amplification Kit (Ambion) with the following modifications: IVT was performed at 37 °C for 16 h. First, strand cDNA was synthesized using SuperScript III (Thermo Fisher Scientific) and a collection of TRAV/TRBV-specific primers. TCRs were then amplified by PCR (20 cycles with the Phusion from New England Biolabs) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR cycle (25 cycles with the Phusion from New England Biolabs) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRα/TCRβ chain. The TCR sequences were further processed using ad hoc Perl scripts to (1) pool all TCR sequences coding for the same protein sequence; (2) filter out all out-frame sequences; and (3) determine the abundance of each distinct TCR sequence. TCR sequences with a single read were not considered for analysis.

Arnaud M., Chiffelle J., Genolet R., Navarro Rodrigo B., Perez M.A., Huber F., Magnin M., Nguyen-Ngoc T., Guillaume P., Baumgaertner P., Chong C., Stevenson B.J., Gfeller D., Irving M., Speiser D.E., Schmidt J., Zoete V., Kandalaft L.E., Bassani-Sternberg M., Bobisse S., Coukos G, & Harari A. (2021). Sensitive identification of neoantigens and cognate TCRs in human solid tumors. Nature Biotechnology, 40(5), 656-660.

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Quantifying m6A RNA Methylation

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Total RNA and poly (A)⁺ RNA methylation were quantified using the m⁶A RNA Methylation Colorimetric Quantification Kit from Abcam (#ab185912) and EpiGentek (EpiQuik m6A RNA Methylation Quantification Kit; #P-9005), respectively, as per the manufacturer’s protocol. Relative m⁶A RNA methylation status was then calculated using the manufacturer’s supplied formula. Poly (A)⁺ RNA was isolated using the Dynabeads mRNA Direct Purification Kit (#61012, Life Technologies).

Panneerdoss S., Eedunuri V.K., Yadav P., Timilsina S., Rajamanickam S., Viswanadhapalli S., Abdelfattah N., Onyeagucha B.C., Cui X., Lai Z., Mohammad T.A., Gupta Y.K., Huang T.H., Huang Y., Chen Y, & Rao M.K. (2018). Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression. Science Advances, 4(10), eaar8263.

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High-throughput RNA-seq Protocol

Cited 2 times

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RNA-seq was performed as described previously.^{45 (link)} cDNA libraries were prepared using Dynabeads mRNA DIRECT Purification Kit (Life Technologies) and Ion Total RNA-Seq Kit v.2 (Life Technologies). High-throughput sequencing of the cDNA fragments was performed using Ion PROTON, Ion PI Template OT2 200 Kit v.3 and Ion PI Sequencing 200 Kit v.3 or Ion PI IC 200 Kit (Life Technologies) following the manufacturer's protocols. P1 chip v.2 (Life Technologies) was used to sequence three pooled barcoded samples. Sequence reads, whose individual read lengths were determined by evaluating the default sequencing quality using the Torrent Server (Life Technologies), were aligned against the human reference transcriptome (NCBI Build 37, hg19) in TopHat2 (http://ccb.jhu.edu/software/tophat/index.shtml).^{46 (link)} Expression levels were calculated by using the cuffdiff function of Cufflinks (http://cufflinks.cbcb.umd.edu/). Raw sequencing data with FPKM (fragments per kilobase of exon per million mapped sequence reads) calculation results are available at GEO (GSE66741 and GSE60559).

Mizutani A., Koinuma D., Seimiya H, & Miyazono K. (2015). The Arkadia-ESRP2 axis suppresses tumor progression: analyses in clear-cell renal cell carcinoma. Oncogene, 35(27), 3514-3523.

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RNA Isolation and qPCR Analysis

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Total oocyte RNA was isolated using a Dynabeads® mRNA DIRECT™ Purification Kit (Life Technologies Lot. USA), according to the manufacturer’s instruction. A first cDNA strand was synthesized using a Reverse Transcription System A3500 (Promega, USA). Real-Time quantitative PCR was done using SYBR Premix Ex Taq (Takara) in a reaction volume of 20 μL and conducted using a fast real-time PCR system (ABI Step One Plus, Life Technology Lot. USA). Primers were synthesized by Invitrogen Lot. Primer sequences are given in Supporting Information (Supporting Information Table S1). Data were analyzed by ΔΔCt method, and the GAPDH was used as the housekeeper gene.

Hou Y.J., Zhu C.C., Duan X., Liu H.L., Wang Q, & Sun S.C. (2016). Both diet and gene mutation induced obesity affect oocyte quality in mice. Scientific Reports, 6, 18858.

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Bulk RNA-seq Analysis of Dendritic Cells

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For bulk RNA-seq analysis, 5,000 DC were facs-sorted into 1.7 ml LoBind microtubes (Eppendorf) containing 40 μl of lysis buffer (Life Technologies). RNA was captured with Dynabeads mRNA Direct Purification Kit (Life Technologies) according to the manufacturer's instructions. The RNA-seq protocol for the generation of libraries is a derivation of MARS-seq (36 (link)). RNA-seq libraries were sequenced using Illumina NexSeq-500, raw data were mapped to the genome (NCBI37/mm9) using HISAT (version 0.1.6) (57 (link)), only reads with unique mapping were considered. Gene expression levels were calculated using HOMER software package (analyzeRepeats.pl rna mm9 -d < tagDir> -count exons -condenseGenes -strand + -raw) (58 (link)). Normalization and differential expression analysis were done using the DESeq2 R-package (Bioconductor, https://bioconductor.org/packages/release/bioc/html/DESeq2.html) (59 (link)). Differentially expressed genes were selected using a 2-fold change and p-value < 0.05 (Figures 3B,D), or a 1.8-fold-change and p-value < 0.05 (Figures 4B,C) or p-value < 0.01(Figure 4D) between at least two conditions. Gene expression matrix was clustered using a k-means algorithm (Matlab function kmeans) with correlation as the distance metric. Heat maps were generated using Genee software.

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Maize Seedling RNA Extraction

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Total RNA and mRNA were extracted from aphid-infested and non-infested (control) maize seedlings. The freshly collected leaves were ground in liquid nitrogen with the use of sterile ceramic mortar and pestle. Isolation of total RNA from maize seedling leaves was carried out using Spectrum Plant Total RNA Kit (Sigma-Aldrich, Poznań, Poland; catalogue no. STRN50), and traces amount of genomic DNA was cleaved with On-Column DNase I Digestion Set (Sigma-Aldrich; catalogue no. DNASE70). Extraction of mRNA was achieved with application of Dynabeads mRNA DIRECT Purification Kit (Life Technologies, Warsaw, Poland; catalogue no. 61012). Isolation procedures were performed, according to the manufacturers’ protocols. Quantity and purity of RNA was evaluated using the Epoch UV-Vis microplate spectrophotometer (BioTek). Only intact and high-quality RNA samples were included in the study.

Sytykiewicz H., Łukasik I., Goławska S, & Chrzanowski G. (2019). Aphid-Triggered Changes in Oxidative Damage Markers of Nucleic Acids, Proteins, and Lipids in Maize (Zea mays L.) Seedlings. International Journal of Molecular Sciences, 20(15), 3742.

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Quantitative Real-Time PCR Analysis of Gene Expression

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For analysis of gene expression by quantitative real-time PCR, total RNA was extracted from cecal tissue with TRI Reagent (Molecular Research Center; Cincinnati, OH). RNA from DSS-treated mice was further purified using the Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to manufacturer recommendations. Reverse transcription reagents (Roche) were employed to generate cDNA from all RNA samples. Real-time PCR was performed using SYBR Green (Roche, Indianapolis, IN) and the Roche Lightcycler 480 Instrument II system (Roche, Indianapolis, IN). Data were analyzed using the comparative delta-delta-Ct method. Target gene transcription of each tissue sample was normalized to the respective levels of Actb mRNA (β-actin). For qPCR analysis of bacterial transcripts, transcription of mcmA and mchB was normalized to bacterial gapA mRNA levels. Data represents at least three independent experiments. DNA contamination was less than 1% for all bacterial amplicons, as determined by separate mock reactions lacking reverse transcriptase. All primers used are listed in Supplementary Table 6.

Sassone-Corsi M., Nuccio S.P., Liu H., Hernandez D., Vu C.T., Takahashi A.A., Edwards R.A, & Raffatellu M. (2016). Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature, 540(7632), 280-283.

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RNA Extraction and qRT-PCR Protocols

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For cell sorting experiments, RNA was extracted using the RNeasy micro kit (QIAGEN, #74004) . RNA of sorted cells from MIP-GFP mice used for RNA sequencing experiment, was extracted using Dynabeads mRNA DIRECT Purification Kit (Life Technologies, #61011). For all other experiments, cells were lysed using Tri-reagent (Sigma, T9424) and RNA was extracted using the Direct-zol RNA miniprep kit (Zymo research, #R2052).
cDNA synthesis and quantitative real-time PCR 100-1000 ng of RNA was used for cDNA synthesis using SensiFast cDNA synthesis kit (Bioline, #BIO-65054). qRT-PCR was carried using Power SYBR green PCR mix (Applied Biosystems, #4367660) using an Applied Biosystems 7300 real time PCR system. Relative transcript levels were calculated using the standard curve method. All results were normalized to the relative expression levels of the house-keeping genes HPRT or TBP. Primer sequences for qRT-PCR are listed in Table S4.

Elhanani O., Salame T.M., Sobel J., Leshkowitz D., Povodovski L., Vaknin I., Kolodkin-Gal D, & Walker M.D. (2020). REST Inhibits Direct Reprogramming of Pancreatic Exocrine to Endocrine Cells by Preventing PDX1-Mediated Activation of Endocrine Genes. Cell reports, 31(5).

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Single-Cell mRNA Purification and Barcoding

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500–20,000 sorted cells were lysed with 100 µl of lysis/binding buffer (Life Technologies), snap-frozen on dry ice and stored at –80 °C until further use. mRNA purification was performed with the Dynabeads mRNA DIRECT Purification Kit (Life Technologies) according to the manufacturer’s guidelines. MARS-seq barcoded RT primers were used for reverse transcription with the Affinity Script cDNA Synthesis Kit (Agilent) in a 10 µl reaction volume.

Lyras E.M., Zimmermann K., Wagner L.K., Dörr D., Klose C.S., Fischer C., Jung S., Yona S., Hovav A.H., Stenzel W., Dommerich S., Conrad T., Leutz A, & Mildner A. (2022). Tongue immune compartment analysis reveals spatial macrophage heterogeneity. eLife, 11, e77490.

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