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Nucleospin rna plus

Manufactured by Takara Bio
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Sourced in Japan, United States

NucleoSpin RNA Plus is a laboratory equipment product designed for the purification of high-quality RNA from a variety of sample types. It utilizes a silica membrane-based technology to efficiently capture and purify RNA molecules.

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Lab products found in correlation

Primescript rt master mix, by Takara Bio (9 mentions) Primescript rt reagent kit, by Takara Bio (5 mentions) Lightcycler 480, by Roche (3 mentions) High capacity rna cdna kit, by Thermo Fisher Scientific (3 mentions) Comparative ct method, by Thermo Fisher Scientific (3 mentions) Thunderbird next sybr qpcr mix, by Toyobo (3 mentions) Lightcycler 480 instrument, by Roche (2 mentions) Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions) Truseq stranded rna lt kit, by Illumina (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Sybr fast qpcr kit, by Roche (2 mentions) Primescript 2 high fidelity rt pcr kit, by Takara Bio (2 mentions) Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions) Kod plus ver 2, by Toyobo (2 mentions) Quantseq 3 end labeling kit, by Lexogen (2 mentions) Thunderbird sybr qpcr mix, by Toyobo (2 mentions) Thermal cycler dice real time system, by Takara Bio (2 mentions) Sybr premix ex taq, by Takara Bio (2 mentions) Random primer, by Thermo Fisher Scientific (2 mentions) Superscript 3 rt, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

NucleoSpin RNA (111 citations) NucleoSpin RNA Plus kit (57 citations) NucleoSpin RNA Plus XS (6 citations) NucleoSpin RNA Plus XS kit (6 citations)

51 protocols using nucleospin rna plus

1

Quantitative Analysis of RNA Expression

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Total RNA was extracted from the transfected cells 72 hours post-transfection using the NucleoSpin® RNA Plus (Clontech, cat# 740984.250), and 250 ng of purified RNA was used for cDNA synthesis using High-Capacity RNA-cDNA kit (ThermoFisher, cat# 4387406). cDNA was diluted 1:20 and 3 ul of cDNA was used for quantitative PCR (qPCR). qPCR reaction samples were prepared using cDNA, SYBR (ThermoFisher, cat# 4385612), and primers detecting each target transcript. Primer sequences are listed in Supplementary Table 2. qPCR was performed using Roche LightCycler480 with the following cycling protocols (Supplementary Table 2). When Ct values are over 35, we considered them as 35, because Ct values fluctuate for very low expressed transcripts. Samples that were transfected with LbCpf1 crRNA backbone plasmid, BPK3082 were used as negative controls, and the levels of fold activation over negative controls were normalized to the expression of HPRT1.
Tak Y.E., Kleinstiver B.P., Nuñez J.K., Hsu J.Y., Horng J.E., Gong J., Weissman J.S, & Joung J.K. (2017). Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors. Nature methods, 14(12), 1163-1166.
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2

Quantitative Gene Expression Analysis

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For gene expression assays, total RNA was isolated from cells using NucleoSpin RNA Plus (Clontech). cDNA was obtained using a high-capacity RNA to-cDNA kit (Applied Biosystems). 500 nanograms of template total RNA and random hexamers were used for each reaction. Real-time PCR amplification was performed using fast SYBR Green Master Mix (Life Technologies) and specific PCR primers in a Lightcycler 480 instrument (Roche). Oligonucleotides used are provided in Table S1. Relative quantification of each target, normalized to an endogenous control (GAPDH or HPRT1), was performed using the comparative Ct method (Applied Biosystems). Error bars indicate SD of three technical replicates and represent at least two independent biological experiments. Statistical analyses were performed by Student’s t test. In heatmaps, log2 qRT-PCR expression values were averaged across biological replicates in each condition and scaled for each gene.
Boulay G., Sandoval G.J., Riggi N., Iyer S., Buisson R., Naigles B., Awad M.E., Rengarajan S., Volorio A., McBride M.J., Broye L.C., Zou L., Stamenkovic I., Kadoch C, & Rivera M.N. (2017). Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain. Cell, 171(1), 163-178.e19.
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3

Gene Expression Analysis Protocol

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For gene expression assays, total RNA was isolated from cells using NucleoSpin RNA Plus (Clontech). cDNA was obtained using SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific). One microgram of template total RNA and random hexamers were used for each reaction. Real-time PCR amplification was performed using fast SYBR Green master mix (Life Technologies) and specific PCR primers in a LightCycler 480 instrument (Roche). The oligonucleotides used are shown in Supplemental Table S3. Relative quantification of each target, normalized to an endogenous control (GAPDH or HPRT1), was performed using the comparative Ct method (Applied Biosystems). Error bars indicate the standard deviation of three technical replicates and represent at least two independent biological experiments. Statistical analyses were performed by Student's t-test. In heat maps (Supplemental Fig. S4B), log2 RT-qPCR expression values were averaged across biological replicates in each condition.
Boulay G., Volorio A., Iyer S., Broye L.C., Stamenkovic I., Riggi N, & Rivera M.N. (2018). Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies. Genes & Development, 32(15-16), 1008-1019.
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4

Illumina RNA-seq Library Preparation

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Total RNA was isolated from cells using NucleoSpin RNA Plus (Clontech). 0.5–1 μg of total RNA was treated with Ribogold zero to remove ribosomal RNA. Illumina sequencing libraries were constructed using random primers according to the manufacturer’s instructions using the Truseq Stranded RNA LT Kit.
Boulay G., Cironi L., Garcia S.P., Rengarajan S., Xing Y.H., Lee L., Awad M.E., Naigles B., Iyer S., Broye L.C., Keskin T., Cauderay A., Fusco C., Letovanec I., Chebib I., Nielsen P.G., Tercier S., Cherix S., Nguyen-Ngoc T., Cote G., Choy E., Provero P., Suvà M.L., Rivera M.N., Stamenkovic I, & Riggi N. (2020). The chromatin landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms and dependencies. Life Science Alliance, 4(2), e202000808.
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5

Quantitative Analysis of RNA Expression

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Total RNA was extracted from the transfected cells 72 hours post-transfection using the NucleoSpin® RNA Plus (Clontech, cat# 740984.250), and 250 ng of purified RNA was used for cDNA synthesis using High-Capacity RNA-cDNA kit (ThermoFisher, cat# 4387406). cDNA was diluted 1:20 and 3 ul of cDNA was used for quantitative PCR (qPCR). qPCR reaction samples were prepared using cDNA, SYBR (ThermoFisher, cat# 4385612), and primers detecting each target transcript. Primer sequences are listed in Supplementary Table 2. qPCR was performed using Roche LightCycler480 with the following cycling protocols (Supplementary Table 2). When Ct values are over 35, we considered them as 35, because Ct values fluctuate for very low expressed transcripts. Samples that were transfected with LbCpf1 crRNA backbone plasmid, BPK3082 were used as negative controls, and the levels of fold activation over negative controls were normalized to the expression of HPRT1.
Tak Y.E., Kleinstiver B.P., Nuñez J.K., Hsu J.Y., Horng J.E., Gong J., Weissman J.S, & Joung J.K. (2017). Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors. Nature methods, 14(12), 1163-1166.
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6

Quantitative Real-Time PCR Analysis of mRNA Expression

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Total RNA was extracted from the transfected cells 72 h post-transfection using the NucleoSpin RNA Plus (Clontech), and 250 ng of purified RNA was used for cDNA synthesis in 20ul of total reaction using High-Capacity RNA-cDNA kit (ThermoFisher). cDNA was diluted 1:20 and 3 μL of cDNA was used for qPCR using SYBR Green Real-Time PCR Master Mix (ThermoFisher), and primers specific for the target transcript (Table S5). qPCR was performed using Roche LightCycler480 with the following cycling protocols: initial denaturation at 95°C for 20 s (s) followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Ct values over 35, were considered as 35. Relative quantification of each target, normalized to an endogenous control (GAPDH or HPRT1), was performed using the comparative Ct method (Applied Biosystems).
Tak Y.E., Boulay G., Lee L., Iyer S., Perry N.T., Schultz H.T., Garcia S.P., Broye L., Horng J.E., Rengarajan S., Naigles B., Volorio A., Sander J.D., Gong J., Riggi N., Joung J.K, & Rivera M.N. (2022). Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors. Cell Genomics, 2(4), 100119.
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7

Depleting Ribosomal RNA for RNA-Seq

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Total RNA was isolated from cells using NucleoSpin RNA Plus (Clontech). 0.5–1 ug of total RNA was treated with Ribogold zero to remove ribosomal RNA. Illumina sequencing libraries were constructed using random primers according to the manufacturer’s instructions using the Truseq Stranded RNA LT Kit.
Boulay G., Sandoval G.J., Riggi N., Iyer S., Buisson R., Naigles B., Awad M.E., Rengarajan S., Volorio A., McBride M.J., Broye L.C., Zou L., Stamenkovic I., Kadoch C, & Rivera M.N. (2017). Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain. Cell, 171(1), 163-178.e19.
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8

RNA-Seq Analysis Pipeline for Differential Gene Expression

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Total RNA was isolated from cells using NucleoSpin RNA Plus (Clontech). For Figure 2H, RNA libraries were prepared from 500 ng of total RNA treated with Ribogold zero to remove rRNA using TruSeq Stranded Total RNA Library Prep Gold kit (Illumina, 20,020,599) and TruSeq RNA Single Indexes. The RNA libraries were sequenced with PE 32 cycles on an Illumina Nextseq500 system. For Figure 3G, RNA samples were sent to Novogene Corporation for mRNA sequencing. RNA libraries were sequenced with PE150 cycles on an Illumina NovaSeq 6000 system. Reads were aligned to hg19 using STAR.59 (link) Mapped reads were filtered to exclude PCR duplicates and reads mapping to known rRNA coordinates, obtained from the rmsk table in the UCSC database (http://genome.ucsc.edu). Gene expression was calculated using featureCounts.60 (link) Only primary alignments with mapping quality of 10 or more were counted. Counts were then normalized to one million reads. Signal tracks were generated using bedtools.56 (link) Differential expression was calculated using DESeq2.61 (link)
Tak Y.E., Boulay G., Lee L., Iyer S., Perry N.T., Schultz H.T., Garcia S.P., Broye L., Horng J.E., Rengarajan S., Naigles B., Volorio A., Sander J.D., Gong J., Riggi N., Joung J.K, & Rivera M.N. (2022). Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors. Cell Genomics, 2(4), 100119.
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9

Quantitative RT-qPCR Analysis of Gene Expression

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Total RNA isolation was performed using NucleoSpin RNA Plus (Takara Bio), after which RNA concentrations were measured using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). A total of 500 ng of RNA was used for reverse transcription with the PrimeScript RT reagent kit (Takara Bio). cDNA templates were subjected to qPCR analysis using the TaqMan Gene Expression Assay Mastermix and TaqMan probes (Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The probe list for the TaqMan gene expression assays is shown in Supplementary Table 5. Each amplification reaction was performed in triplicate, and the average of three threshold cycles was used to calculate the relative amount of transcript in the sample (StepOne Software; Thermo Fisher Scientific). mRNA was quantified and expressed in arbitrary units as the ratio of the sample quantity to the calibrator or the mean values of control samples. All values were normalized to an endogenous control, the human β-actin gene.
Abe Y., Sano T., Otsuka N., Ogawa M, & Tanaka N. (2024). PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth. Communications Biology, 7, 593.
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10

Real-Time qPCR Analysis of Gene Expression

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Total RNA was extracted using NucleoSpin® RNA Plus (Takara Bio, Shiga, Japan) and cDNA was synthesized using PrimeScript™ RT Master Mix (Perfect Real Time; Takara Bio) according to the respective manufacturer’s instructions. Real-time PCR was performed using a TaKaRa PCR Thermal Cycler Dice® (Takara Bio) and SYBR Fast qPCR kit (Kapa Biosystems, Cape Town, South Africa) using the following amplification protocol: initial incubation at 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min, followed by melting curve analysis. Gene expression was calculated from relative standard curves, normalized to GAPDH, and analyzed using the 2−ΔΔCT method43 (link). The following primer sequences were used: HER3 (F: 5′-TGC TGA GAA CCA ATA CCA GAC A-3′, R: 5′-CTG TCA CTT CAC GAA TCC ACT G-3′); NRG1 (F: 5′-AGT CCT TCG GTG TGA AAC CAG-3′, R: 5′-TGC GAA GTT CTG ACT TCC CTG-3′); E-cadherin (F: 5′-GAA CGC ATT GCC ACA TAC AC-3′, R: 5′-GAA TTC GGG CTT GTT GTC AT-3′); and GAPDH (F: 5′-CTG CAC CAA CTG CTT AG-3′, R: 5′-TGA AGT CAG AGG AGA CCA CC-3′).
Tanimura K., Yamada T., Okada K., Nakai K., Horinaka M., Katayama Y., Morimoto K., Ogura Y., Takeda T., Shiotsu S., Ichikawa K., Watanabe S., Morimoto Y., Iwasaku M., Kaneko Y., Uchino J., Taniguchi H., Yoneda K., Matoba S., Sakai T., Uehara H., Yano S., Kusaba T., Katayama R, & Takayama K. (2022). HER3 activation contributes toward the emergence of ALK inhibitor-tolerant cells in ALK-rearranged lung cancer with mesenchymal features. NPJ Precision Oncology, 6, 5.
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