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Rneasy mini kit with rnase free dnase set

Manufactured by Qiagen
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The RNeasy Mini Kit with RNase-Free DNAse Set is a lab equipment product designed for the purification of total RNA from various sample types. The kit utilizes a silica-based membrane technology to provide efficient RNA isolation. The included RNase-Free DNAse Set allows for the removal of genomic DNA contamination during the purification process.

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Close spelling variants of this product

RNeasy Mini Kit (41418 citations) RNeasy kit (11240 citations) RNase-Free DNase Set (2366 citations) RNase-free DNase (842 citations) DNase (494 citations) RNeasy Mini (299 citations) RNase-free DNase kit (281 citations) RNase mini kit (96 citations) DNase set (33 citations) RNase-Free DNase Set kit (20 citations)

6 protocols using rneasy mini kit with rnase free dnase set

1

Isolation and m6A Enrichment of mRNA

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Total RNA from cultured fetal liver cells was isolated using TRIzol followed by RNeasy Mini Kit with RNase-Free DNase Set (QIAGEN). Then poly(A)+ mRNA was isolated from 3 to 5 μg of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Then the isolated RNAs were denatured at 65 °C for 5 min, cooled on ice, and used for m6A-IP using EpiMark N6-Methyladenosine Enrichment Kit (New England Biolabs) according to the manufacturer’s protocol. IP and input samples were used for cDNA synthesis and qPCR analysis as described above. mRNA expression levels of genes were normalized to Gapdh.
Yoshinaga M., Han K., Morgens D.W., Horii T., Kobayashi R., Tsuruyama T., Hia F., Yasukura S., Kajiya A., Cai T., Cruz P.H., Vandenbon A., Suzuki Y., Kawahara Y., Hatada I., Bassik M.C, & Takeuchi O. (2022). The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity. Nature Communications, 13, 6435.
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2

Quantifying Developmental Gene Expression

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Total RNA was extracted from FL and HL bud samples using the RNeasy Mini Kit with RNase-Free DNAse Set and a TissueLyser LT (Qiagen). For each sample, a cDNA library was prepared using M-MLV Reverse Transcriptase with oligo(dT) primer (ThermoFisher Scientific). Intron-spanning amplicons from pigeon PITX1, TBX5, and ACTB were amplified from cDNA libraries using a CFX96 instrument and iTaq Universal Sybr Green Supermix (BioRad). Primer sequences were published previously (Domyan et al., 2016 (link)). For a single qRT-PCR experiment, three technical replicates were performed for each sample and the mean value was determined. Each experiment was repeated three times. Statistical significance was determined using a pairwise Wilcoxon rank sum test in R version 3.3.3 (R Core Team, 2018 ).
Boer E.F., Van Hollebeke H.F., Park S., Infante C.R., Menke D.B, & Shapiro M.D. (2019). Pigeon foot feathering reveals conserved limb identity networks. Developmental biology, 454(2), 128-144.
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3

RNA-seq Library Preparation and Sequencing

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To prepare libraries from the cells for RNA‐seq, an RNeasy Mini Kit with RNase‐Free DNase Set (QIAGEN), Dynabeads mRNA DIRECT Micro Purification Kit, and Ion Total RNA‐Seq Kit v2 (Thermo Fisher Scientific) were used according to the manufacturers' protocols. cDNA libraries were quantified using an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). Sequencing was performed with an Ion Proton System (Thermo Fisher Scientific) using an Ion PI Hi‐Q Chef Kit (Thermo Fisher Scientific) and Ion PI Chip Kit v3 (A26770, Thermo Fisher Scientific). Reads were aligned to the human genome (Genome Reference Consortium Human Build 37 (GRCh37)/hg19) as a reference genome using TopHat2 open‐source bioinformatics tool. Fragments per kilobase of exon per million sequence reads (FPKM) values were calculated using the Cuffdiff function of Cufflinks. Raw sequence data have been deposited in the Gene Expression Omnibus (GEO) database (accession number GSE152414).
Tamura Y., Morikawa M., Tanabe R., Miyazono K, & Koinuma D. (2021). Anti‐pyroptotic function of TGF‐β is suppressed by a synthetic dsRNA analogue in triple negative breast cancer cells. Molecular Oncology, 15(5), 1289-1307.
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4

Beak Development in Pigeon Embryos

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Pigeon eggs were collected from Racing Homer (medium beak) and Oriental Frill (short beak) breeding pairs and incubated to embryonic day 6 (Hamburger-Hamilton (HH) stage 28–29,60 (link)). Facial prominences that form the upper beak (frontonasal and maxillary, FNP+MXP) and lower beak (mandibular, MDP) were dissected and stored separately in RNAlater (ThermoFisher Scientific) at −80°C. Additional tissue was harvested from each embryo and used for DNA extraction and sex determination following a previously published PCR-based assay61 . Total RNA was extracted from embryonic tissue samples using the RNeasy Mini Kit with RNase-Free DNAse Set and a TissueLyser LT (Qiagen).
Boer E.F., Van Hollebeke H.F., Maclary E.T., Holt C., Yandell M, & Shapiro M.D. (2021). A ROR2 coding variant is associated with craniofacial variation in domestic pigeons. Current biology : CB, 31(22), 5069-5076.e5.
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5

Luciferase Assay and qRT-PCR in HEK293T

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Transient transfection of HEK293T cells was performed with polyethyleneimine (Polysciences) in 12 well plates. Luciferase activity and co-transfected Renilla luciferase activity by pRL-tk (Promega) were subsequently measured with the Dual-Glo Luciferase Kit (Promega) according to the manufacturer's instruction using an ARVO luminometer (Parkin-Elmer). The relative luciferase activity was determined by normalizing the co-transfected Renilla activity.
qRT-PCR analysis qRT-PCR analysis was performed as described previously [44, 45] . Briefly, total RNA was prepared using TRIzol (Invitrogen) or the RNeasy mini kit with RNase-Free DNase Set (QIAGEN), and reverse transcription was performed using the PrimeScript RT Master Mix (Takara Bio). qRT-PCR was performed on an ABI PRISM 7500 or StepOnePlus Real-Time PCR systems (Thermo Fisher Scientific) using TB Green Premix Ex TaqII (Takara Bio). Transcript levels of target mRNA were normalized with rp49 (RpL32; FlyBase) levels in the same samples. Primers used in this study are listed in Table S1.
Nishimura T. (2020). Feedforward Regulation of Glucose Metabolism by Steroid Hormones Drives a Developmental Transition in Drosophila. Current biology : CB, 30(18).
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6

Pigeon Craniofacial Development Protocol

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Pigeon eggs were collected from Racing Homer (medium beak) and Oriental Frill (short beak) breeding pairs and incubated to embryonic day 6 (Hamburger-Hamilton (HH) stage 28-29, 58 ).
Facial prominences that form the upper beak (frontonasal and maxillary, FNP+MXP) and lower beak (mandibular, MDP) were dissected and stored separately in RNAlater (ThermoFisher Scientific) at -80°C. Additional tissue was harvested from each embryo and used for DNA extraction and sex determination following a previously published PCR-based assay 59 . Total RNA was extracted from embryonic tissue samples using the RNeasy Mini Kit with RNase-Free DNAse Set and a TissueLyser LT (Qiagen).
Boer E.F., Hollebeke H.F.V., Holt C., Yandell M., & Shapiro M.D. (2021). A ROR2 coding variant is associated with craniofacial variation in domestic pigeons.
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