Nucleospin kit

Manufactured by Takara Bio

Sourced in Japan

The NucleoSpin kit is a nucleic acid purification system designed for the efficient extraction and isolation of DNA, RNA, or plasmids from a variety of sample sources. The kit utilizes a silica-membrane technology to provide reliable and consistent results.

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Lab products found in correlation

Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Quantitect sybr 1 kit, by Qiagen (2 mentions) Kod fx kit, by Toyobo (2 mentions) Quantitect reverse transcription kit, by Qiagen (2 mentions) Pgem t vector, by Promega (1 mentions) Hiseq 2000 sequencer, by Illumina (1 mentions) Clc genomics workbench software, by Qiagen (1 mentions) Nebnext ultra rna library prep kit, by New England Biolabs (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Lightcycler 96 system, by Roche (1 mentions) One step sybr primescript plus rt pcr kit, by Takara Bio (1 mentions) Quantitect reverse transcription kit, by Toyobo (1 mentions) Bsiwi, by New England Biolabs (1 mentions) Truseq stranded total rna library prep kit, by Illumina (1 mentions) Miseq instrument, by Illumina (1 mentions) Ribo zero human kit, by Illumina (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Tb green premix ex taq 2, by Takara Bio (1 mentions) Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions) Revertra ace, by Toyobo (1 mentions)

Close spelling variants of this product

NucleoSpin Gel and PCR Clean-up kit (92 citations) NucleoSpin Tissue kit (56 citations) NucleoSpin RNA XS kit (39 citations) NucleoSpin® RNA isolation kit (19 citations) NucleoSpin PCR Clean-up Kit (9 citations) Nucleospin RNA extraction kit (9 citations) NucleoSpin DNA Stool kit (5 citations) NucleoSpin microRNA isolation kit (4 citations) NucleoSpin TriPrep kit (4 citations) NucleoSpin miRNA kit (3 citations)

17 protocols using nucleospin kit

Cloning and Sequencing of SMN Transcripts

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The PCR product of SMN transcript was purified and cloned into a TA cloning vector, pGEM-T vector (Promega, Madison, WI, USA), before being transformed into competent Escherichia coli (Invitrogen, Carlsbad, CA) and propagated in LB solution. The transformed E. coli was grown on an LB plate containing IPTG and X-gal for blue/white colony selection.
The white colonies were selected and subjected to PCR using T7-SP6 primer set and were run on an agarose gel to select the target colonies based on the amplified product size. The PCR product corresponding to cryptic-exon (exon 7a)-containing transcripts was then purified using Nucleospin kit, in accordance with the manufacturer’s protocol (Takara Bio, Tokyo, Japan), and subjected to direct sequencing analysis. This sequencing analysis was outsourced to Fasmac Co. Ltd.

Wijaya Y.O., Niba E.T., Nishio H., Okamoto K., Awano H., Saito T., Takeshima Y, & Shinohara M. (2022). High Concentration or Combined Treatment of Antisense Oligonucleotides for Spinal Muscular Atrophy Perturbed SMN2 Splicing in Patient Fibroblasts. Genes, 13(4), 685.

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RNA-seq Analysis of Evi1-Overexpressing Bone Marrow Cells

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Total RNA was isolated from GFP^pos, lineage^neg, c‐kit^pos Evi1‐overexpressing or the control GFP^pos, lineage^neg, c‐kit^pos bone marrow mononuclear cells by using a NucleoSpin kit (Takara Bio). The quality of the RNA samples (RNA Integrity Number >8) was validated using a Agilent 2100 Bioanalyzer (Agilent Technology). For RNA library preparation, a NEB Next Ultra RNA Library Prep Kit (New England Biolabs) was used. RNA library samples were sequenced according to the manufacturer's protocol using an Illumina Hiseq 2000 sequencer (Illumina). The sequence data obtained were analyzed using CLC Genomics Workbench software (Qiagen) and R software (http://www.R‐project.org).

Mizuno H., Koya J., Masamoto Y., Kagoya Y, & Kurokawa M. (2021). Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation. Cancer Science, 112(10), 4112-4126.

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BRSV Infection and PM Exposure in A549 Cells

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A549 cells were infected with BRSV for 3 days and then exposed to PM for 60 min. Total RNA was extracted from the cell lysates using a NucleoSpin kit (TaKaRa, Kyoto, Japan). Quantitative RT-PCR (qRT-PCR) was performed using a One Step SYBR PrimeScript plus RT-PCR kit (TaKaRa, Kyoto, Japan). The primer sets and amplification conditions for qRT-PCR of IL-1β, IL-6, IL-8, MCP-1, and RANTES mRNA were described previously (Lau et al., 2013 (link)). Data were normalized to expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Amplification was carried out in a LightCycler 96 system (Roche, CT, USA). All experiments were performed in duplicate. Relative expression of mRNA between infected samples and uninfected controls was calculated using the 2^−ΔΔCT method and expressed as a -fold change.

Sudaryatma P.E., Nakamura K., Mekata H., Sekiguchi S., Kubo M., Kobayashi I., Subangkit M., Goto Y, & Okabayashi T. (2018). Bovine respiratory syncytial virus infection enhances Pasteurella multocida adherence on respiratory epithelial cells. Veterinary Microbiology, 220, 33-38.

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Quantitative Real-Time PCR Analysis of Canine BC Stem Cell Markers

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Quantitative real-time PCR was performed as described previously^{14 (link)}. Total RNA was extracted from 2 × 10⁵ of 2.5D organoid cells at early and late passage, 3D organoids, and urothelial carcinoma cell lines by using a NucleoSpin kit (Takara Bio Inc., Shiga, Japan) following the manufacturer’s protocol. First-strand cDNA was prepared using a QuantiTect Reverse Transcription Kit (TOYOBO, Tokyo, Japan). Quantitative real-time PCR was done using a QuantiTect SYBR I Kit (QIAGEN, Hilden, Germany) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The ΔΔCq method was used for quantification. Specific primers used for dog BC stem cell markers, SOX2, CD44, and GAPDH were designed and are shown in Table 3.Table 3

Primers for real-time quantitative PCR analysis.

Gene name	Primer	Sequence
SOX2	Forward	5′-GCCCTGCAGTACAACTCCAT-3′
SOX2	Reverse	5′-GGAGTGGGAGGAGGAGGTAA-3′
CD44	Forward	5′-CCAAGACAGTTCCAGGGTGT-3′
CD44	Reverse	5′-TTGAGGTTTCCGCATAGGAC-3′
GAPDH	Forward	5′-AACTCCCTCAAGATTGTCAGCAA-3′
GAPDH	Reverse	5′-CATGGATGACTTTGGCTAGAGGA-3′

Abugomaa A., Elbadawy M., Yamanaka M., Goto Y., Hayashi K., Mori T., Uchide T., Azakami D., Fukushima R., Yoshida T., Shibutani M., Yamashita R., Kobayashi M., Yamawaki H., Shinohara Y., Kaneda M., Usui T, & Sasaki K. (2020). Establishment of 2.5D organoid culture model using 3D bladder cancer organoid culture. Scientific Reports, 10, 9393.

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Quantitative RT-PCR Analysis Protocol

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Total mRNA was isolated by using the Nucleospin kit (TaKaRa, 740955) according to manufacturer’s instruction. cDNA was synthesized by Super^TM Script^TM VILO cDNA synthesis kit (Thermo Fisher Scientific, 11754050). qPCR was performed by PowerUp^TM SYBR^TM Green Master Mix on QuantStudio 3 or 6. Primer sequences were listed on Supplementary Tables 5 and 6. Data are expressed as a fold-change and were normalized with undifferentiated cells expression.

Kishimoto K., Furukawa K.T., Luz-Madrigal A., Yamaoka A., Matsuoka C., Habu M., Alev C., Zorn A.M, & Morimoto M. (2020). Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nature Communications, 11, 4159.

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DNA Extraction and PCR Amplification

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Total DNA was extracted from cells using the Nucleospin Kit (Takara Bio Inc.). Polymerase chain reaction (PCR) using specific primer sets (Forward: 5′-GTTGAGTTCACGTAGACAGGC-3′, Reverse: 5′-GACTCCCATTCATCATGCCCA-3′) was performed to amplify the DNA using the KOD FX Kit (KOD FX; Toyobo, Osaka, Japan) with the following temperature profile: 94 °C for 2 min, followed by 40 cycles of 98 °C for 10 s and 55 °C for 30 s, and 72 °C for 2 min. The PCR products were treated with the restriction enzyme BsiWI (New England Biolabs, Ipswich, MA, USA). One microgram of DNA was treated with 1 unit of enzyme and NE Buffer 2.1 at 55 °C for 15 min. The samples were analysed by electrophoresis on a 5% polyacrylamide TBE gel.

Taketani Y., Kitamoto K., Sakisaka T., Kimakura M., Toyono T., Yamagami S., Amano S., Kuroda M., Moore T., Usui T, & Ouchi Y. (2017). Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair. Scientific Reports, 7, 16713.

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RNA-seq analysis of canine bladder tissue and cancer models

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Total RNA was extracted from normal bladder tissues from healthy dogs, BC organoid samples and 2D urothelial carcinoma cell lines using the NucleoSpin kit (TaKaRa Bio Inc.) according to the manufacturer's instructions. Total extracted RNA (10 ng) for each sample was used to generate the sequencing libraries. RNA‐seq was performed at the Research and Education Center for Prevention of Global Infectious Disease of Animals, Tokyo University of Agriculture and Technology (Tokyo, Japan). A Ribo‐Zero Human Kit (Illumina) and a TruSeq Stranded Total RNA Library Prep Kit (Illumina) were used for library preparation, followed by sequencing (7.5 million single‐end reads) on an Illumina MiSeq instrument. Initial quality control of RNA‐seq data (FASTQ) for each sample was performed using cutadapt (version 1.8.3) and cmpfastq_pe.pl software. Reads were mapped to the reference genome (CamFam 3.1) using the STAR (version 2.5.1b) software. PCA was performed to display differences between samples. Fragments per kilobase of transcript per million mapped reads were normalized using the trimmed mean of M value method.

Elbadawy M., Usui T., Mori T., Tsunedomi R., Hazama S., Nabeta R., Uchide T., Fukushima R., Yoshida T., Shibutani M., Tanaka T., Masuda S., Okada R., Ichikawa R., Omatsu T., Mizutani T., Katayama Y., Noguchi S., Iwai S., Nakagawa T., Shinohara Y., Kaneda M., Yamawaki H, & Sasaki K. (2019). Establishment of a novel experimental model for muscle‐invasive bladder cancer using a dog bladder cancer organoid culture. Cancer Science, 110(9), 2806-2821.

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Quantifying HBV RNA and DNA Levels

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Culture supernatant DNA was extracted using a NucleoSpin kit (Takara). Total RNA was extracted using RNeasy Mini Kit (QIAGEN), treated with amplification grade DNase I (Thermo Fisher Scientific), and then reverse transcribed into cDNA with a High Capacity cDNA Reverse Transcription Kit (ABI), according to the manufacturer’s instructions. qPCR analysis was performed using TB Green Premix Ex Taq II (Takara) with StepOnePlus Real-Time PCR systems (ABI). Primers sequences are listed in Table S1. Expression of HBV RNA mRNA was normalized to that of the endogenous HPRT. An HBV replicon plasmid was used as a standard to absolutely quantify HBV copy numbers in the culture supernatant.

Li Y., Que L., Fukano K., Koura M., Kitamura K., Zheng X., Kato T., Aly H.H., Watashi K., Tsukuda S., Aizaki H., Watanabe N., Sato Y., Suzuki T., Suzuki H.I., Hosomichi K., Kurachi M., Wakae K, & Muramatsu M. (2020). MCPIP1 reduces HBV-RNA by targeting its epsilon structure. Scientific Reports, 10, 20763.

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Osteopontin Expression in BM-DCs

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Bone marrow cells were collected from femurs and tibias of 2-month-old male or female mice and cultured for 5–7 days in Petri dishes in RPMI1640 medium supplemented with 10% fetal calf serum, antibiotics, and 10 ng/mL GM-CSF to obtain BM-DCs. The BM-DCs were then stimulated with full-length OVA (500 μγ/mL) for 24 h to analyze the transcription level of the osteopontin gene. The effects of hSF on osteopontin expression in BM-DCs were examined by pre-incubating the cells with 10 μL of 10-fold-diluted hSF for 30 min before OVA stimulation. Total RNA was purified form BM-DCs by using a NucleoSpin kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions.

Niikura Y., Ishii T., Murakami J., Narita T., Fujita Y., Negishi H., Taketani Y, & Yamashita N. (2019). Age-related immune-modulating properties of seminal fluid that control the severity of asthma are gender specific. Aging (Albany NY), 11(2), 707-723.

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Transcriptomic Analysis of Bladder Tissues

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Total RNA was extracted from normal bladder tissues, organoid samples, 2D urothelial carcinoma cell lines, and blood samples using the NucleoSpin kit (Takara Bio Inc) according to the manufacturer's instructions. First‐strand cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative real‐time PCR was performed using a QuantiTect SYBR I Kit (QIAGEN) and a StepOnePlus Real‐Time PCR System (Applied Biosystems). The ΔΔCq method was used for quantification. Specific primers used for dog CK5, DSG3, GATA3, ERBB2, MMP28, CTSE, CNN3, TFPI2, COL17A1, AGPAT4, GAPDH, CK15, TGM2, GJB2, IL1R2, COL5A2, CDH3, CHST4, and ADORA2B are listed in Table 2.

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