Nucleospin rna 2

Manufactured by Takara Bio

Sourced in Japan

The NucleoSpin RNA II is a laboratory equipment designed for the isolation and purification of high-quality RNA from a variety of biological samples. It utilizes a silica-membrane technology to efficiently capture and purify RNA molecules.

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34 protocols using nucleospin rna 2

Real-Time PCR Methodology for Gene Expression

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Real-time PCR was performed as previously described (Kojima et al, 2010 (link)). Total RNA was extracted using NucleoSpin RNA Ⅱ (Takara) in accordance with the manufacturer’s protocol. SuperScript II reverse transcriptase (Invitrogen) was also used to synthesize cDNA. The resulting cDNAs were used for PCR using Fast SYBR Green Master Mix (Applied Biosystems) in triplicate. PCR and data collection were performed with a 7500 Fast Real-Time PCR System (Applied Biosystems). Relative gene expressions were analyzed by the ΔΔCt method. Data were normalized relative to the GAPDH or β-actin gene expression level. The primer sequences are listed in Table S6.

Matsumura Y., Ito Y., Mezawa Y., Sulidan K., Daigo Y., Hiraga T., Mogushi K., Wali N., Suzuki H., Itoh T., Miyagi Y., Yokose T., Shimizu S., Takano A., Terao Y., Saeki H., Ozawa M., Abe M., Takeda S., Okumura K., Habu S., Hino O., Takeda K., Hamada M, & Orimo A. (2019). Stromal fibroblasts induce metastatic tumor cell clusters via epithelial–mesenchymal plasticity. Life Science Alliance, 2(4), e201900425.

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Quantitative Real-Time PCR Analysis

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Total RNA was isolated from the cells using Nucleospin RNA Ⅱ (Takara, Shiga, Japan). RNA samples were reverse-transcribed using MuLV reverse transcriptase (Applied Biosystems) in a total volume of 20 μL, and qRT-PCR was performed using the StepOnePlus Real-Time PCR systems (Applied Biosystems). Aliquots of the reverse transcription products were amplified in 20 μL of a reaction mixture containing PowerUp SYBR Green Master Mix (Applied Biosystems) and 0.5 μM each primer. The primer pairs used are listed in Supplementary Table S2.

Makiyama T., Obama T., Watanabe Y, & Itabe H. (2022). Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets. International Journal of Molecular Sciences, 23(12), 6366.

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RNA Extraction and RT-PCR Analysis

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RNA extraction, PCR, RT-PCR and real-time RT-PCR analyses were performed essentially as described previously [28 (link),82 (link)]. The primer sets used in this study were listed in S1 Table. The RNAs from yeast CRY1 treated with or without 2 mM DTT were exacted using NucleoSpin RNA II (Clontech) to obtain cDNAs of IRE1, HAC1 U and HAC1 S.

Zhang L., Chen H., Brandizzi F., Verchot J, & Wang A. (2015). The UPR Branch IRE1-bZIP60 in Plants Plays an Essential Role in Viral Infection and Is Complementary to the Only UPR Pathway in Yeast. PLoS Genetics, 11(4), e1005164.

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Quantifying Stem Cell Differentiation

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The total RNA was isolated from undifferentiated ES and differentiating cells on days 0, 4, 8 and 12 upon differentiation by NucleoSpin RNA II (Clontech). A 2.5 μg of total RNA was converted into cDNA using Maxima H Minus reverse transcriptase (Thermo Scientific) according to the manufacturer's instructions. Real-time PCR was performed on Step One Plus real-time PCR System (Life Technologies) using the Fast SYBR Green Master Mix (Life Technologies). The primers and annealing temperature were: Gapdh (Gapdh-F and Gapdh-R; 60°C); Nanog (Nanog-F and Nanog-R; 60°C); Oct3/4 (Oct3/4-F and Oct3/4-R; 62°C); Xist exon 1–3 (X1) for 129 allele (Xist¹²⁹-E1-3-F and Xist¹²⁹-E1-3-R; 62°C); Xist exon 7 (X2) for 129 allele (Xist¹²⁹-E7-F and Xist¹²⁹-E7-R; 58°C); Tsix intron 3–4 (T1) for 129 (Tsix¹²⁹-In3-4-F and Tsix-In3-4-R; 58°C) and Cast (Tsix^Cast-In3-4-F and Tsix-In3-4-R; 58°C) alleles; Tsix exon 4 (T2) for 129 (Tsix¹²⁹-E4-F and Tsix¹²⁹-E4-R) and Cast (Tsix^Cast-E4-F and Tsix¹²⁹-E4-R; 60°C) alleles; Mecp2 for 129 allele (Mecp2¹²⁹-F and Mecp2¹²⁹-R; 58°C); Pgk1 for 129 allele (Pgk1¹²⁹-F and Pgk1¹²⁹-R; 60°C). The ΔΔCt method was employed to analyze the relative changes in the gene expression levels from the real-time qPCR experiments, and Gapdh was used to normalize the data.

Yamada N., Hasegawa Y., Yue M., Hamada T., Nakagawa S, & Ogawa Y. (2015). Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome. PLoS Genetics, 11(8), e1005430.

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Normalized cDNA Library Construction of Tobacco K326

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Tobacco (K326), provided by the tobacco cultivation and physiology laboratory of Henan Agricultural University, was used in this experiment. A cDNA library of K326 was constructed via a switching mechanism at the 5′ end of RNA transcript (SMART™). Briefly, the root, stem, leaf, and flower of K326 at different developmental stages were quickly frozen in liquid nitrogen and then stored at − 70 °C. Total RNA from different tissues was extracted and mixed together. After purification of total RNA using NucleoSpin RNA II (740,955.20; Clontech, Palo Alto, CA, USA), poly A+ mRNA was enriched using a NucleoTrap mRNA Kit (740,655; Clontech). The first strand of cDNA was then synthesized based on the SMART technique and duplex-specific nuclease (DSN) according to the Matchmaker® Gold Yeast One-Hybrid Library Screening System (Clontech). Double-stranded cDNA was synthesized through long-distance (LD)-PCR. Following purification using a chromatographic column, a normalized cDNA library of K326 was obtained.

Yang H., Zhou Y., Zhang Y., Wang J, & Shi H. (2019). Identification of transcription factors of nitrate reductase gene promoters and NRE2 cis-element through yeast one-hybrid screening in Nicotiana tabacum. BMC Plant Biology, 19, 145.

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RNA-seq Analysis of Sf3b1 Haploinsufficiency in Murine Bone Marrow

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Total RNA was extracted from whole BM of 6-month-old female Sf3b1^+/− (n = 2) and Sf3b1^+/+ (n = 2) mice using NucleoSpin RNA II (Clontech Laboratories). PolyA cDNA was prepared from 3 μg of RNA and mouse RNA-sequencing was run on Illumina HiSeq2000 by Otogenetics (Norcross, GA). 100 basepair paired-end RNA-sequencing reads were mapped to the mm10 RefSeq mouse transcriptome and spliceome by DNAnexus (http://dnanexus.com) using a Bayesian method where a read was mapped when its posterior probability of mapping exceeded 0.9. These filtered posterior probabilities were summed to generate fractional read counts per gene and per exon, with probabilities from splice-junction spanning reads counted for each relevant exon. We used rounded gene and exon read counts as inputs for our differential expression analyses.

Visconte V., Tabarroki A., Zhang L., Parker Y., Hasrouni E., Mahfouz R., Isono K., Koseki H., Sekeres M.A., Saunthararajah Y., Barnard J., Lindner D., Rogers H.J, & Tiu R.V. (2014). Splicing factor 3b subunit 1 (Sf3b1) haploinsufficient mice display features of low risk Myelodysplastic syndromes with ring sideroblasts. Journal of Hematology & Oncology, 7, 89.

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Quantitative Analysis of Cartilage Gene Expression

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Quantitative real-time polymerase chain reaction (rtPCR) was used to assess anabolic and catabolic mRNA expression. Due to known zonal variations in cartilage endogenous gene expression only the superficial zone of each explant was used for analysis ^{29 (link)}. A slice was taken from the top 10–20 % of the tissue to represent the superficial zone of cartilage^{30 (link)}. Total mRNA from this zone was extracted using Nucleospin RNA II (Clontech) according to manufacturer’s protocols. Extracted mRNA from all samples was quantified using Nanodrop 2000 spectrophotometer (Thermo Scientific) and reverse transcribed into cDNA using High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Real-time PCR was performed using Taqman Gene Expression Assays (Applied Biosystems) with the primers for the following genes: GAPDH (Bt03210913_g1), aggrecan (Bt03212186_m1), collagen type II (Bt03251861_m1), MMP-13(Bt03214050_m1) and ADAMTS5(Bt04230783_m1). 40 ng of cDNA template was prepared per 20 μL reaction for the genes of interest and the endogenous gene. Real-time PCR analysis was carried out using a Taqman PCR Master Mix and 7500 Real-Time PCR System (Applied Biosystems). Data was analyzed using the comparative C_T method, where average C_T values were first normalized to GAPDH gene expression followed by relative quantification to the positive control (normal cartilage).

Sharma S., Vazquez-Portalatin N., Calve S, & Panitch A. (2015). Biomimetic molecules lower catabolic expression and prevent chondroitin sulfate degradation in an osteoarthritic ex vivo model. ACS biomaterials science & engineering, 2(2), 241-250.

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Northern Blot Analysis of Transcripts

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Cell fractionation was performed as described previously (20 (link)). Total RNA was extracted (Nucleospin RNAII; Takara Bio) and poly(A)⁺ RNA was purified (NucleoTrap mRNA Mini kit; Takara Bio) according to the manufacturer's instructions. The RNA samples were loaded on 1.5% gels (NorthernMax-Gly Kit; Life Technologies), transferred to Hybond N+ nylon membranes (GE Healthcare) and probed with internally DIG-labeled sequences following pre-hybridization in ULTRAhyb hybridization buffer (Ambion). DIG-labeled RNA probes were prepared from template DNA amplified by specific primers (Supplementary Table S2) using a DIG Northern Starter Kit (Roche). Visualization of transcripts was performed with a CDP-Star reagent (Roche). Signals were detected by an LAS4000 mini biomolecular imager (GE Healthcare). The densitometric analysis of each band was performed by ImageQuant TL analysis software (GE Healthcare).

Koyama A., Sugai A., Kato T., Ishihara T., Shiga A., Toyoshima Y., Koyama M., Konno T., Hirokawa S., Yokoseki A., Nishizawa M., Kakita A., Takahashi H, & Onodera O. (2016). Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43. Nucleic Acids Research, 44(12), 5820-5836.

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mRNA Quantification: Isolation and Measurement

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For mRNA quantification, total RNA was isolated using Nucleospin RNA II (Takara Bio). RNA was quantified with a Nanodrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from 1.5 μg total RNA using PrimeScript RT Master Mix (Takara Bio), and qPCRs were carried out on a StepOnePlus Real‐time PCR system (Thermo Fisher Scientific) using SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio). GAPDH mRNA was used as an internal reference. The primers used in this experiment are listed in Table S3. Relative transcript levels were calculated with the 2^−ΔΔCT method.

Mori A., Masuda K., Ohtsuka H., Shijo M., Ariake K., Fukase K., Sakata N., Mizuma M., Morikawa T., Hayashi H., Nakagawa K., Motoi F., Naitoh T., Fujishima F, & Unno M. (2018). FBXW7 modulates malignant potential and cisplatin‐induced apoptosis in cholangiocarcinoma through NOTCH1 and MCL1. Cancer Science, 109(12), 3883-3895.

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Total RNA Extraction and RT-qPCR Analysis

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Total RNA was extracted with NucleoSpin RNAII (Takara Bio, Otsu, Japan), and RT was performed with PrimeScript^® RT Master Mix (Takara Bio). The PCR was set up with SYBER Premix Ex Taq^™ II (Takara Bio) and carried out on a Thermal Cycler Dice Real Time System II device (Takara Bio). The sequences of the specifically designed primers are listed in Table 1.

Kitazawa M., Hida S., Fujii C., Taniguchi S., Ito K., Matsumura T., Okada N., Sakaizawa T., Kobayashi A., Takeoka M, & Miyagawa S.I. (2017). ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication. PLoS ONE, 12(1), e0169340.

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