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Nucleospin rna 2 kit

Manufactured by Takara Bio
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Sourced in Japan, United States

The NucleoSpin RNA II kit is a laboratory equipment designed for the efficient isolation and purification of total RNA from various biological samples, including cells, tissues, and bacteria. The kit utilizes a silica-membrane technology to selectively bind RNA, allowing for effective removal of contaminants and the subsequent elution of high-quality RNA.

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Spelling variants of this product

NucleoSpin RNA kit (154 citations) NucleoSpin RNA (111 citations) NucleoSpin RNA II (34 citations) NucleoSpin kit (17 citations) NucleoSpin II (2 citations)

65 protocols using nucleospin rna 2 kit

1

Quantitative RT-PCR for Gene Expression

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Total RNA was isolated from YAMC and HCT116 cells using the NucleoSpin RNA II kit (Clontech Laboratories, Mountain View, CA, USA). PrimeTime® qPCR primers for murine Gsta4, human GSTA4, JUN, Nrf2, and β-actin were purchased from Integrated DNA Technologies. qRT-PCR was performed using iTaq™ universal SYBR® Green one-step kit (Bio-Rad, Hercules, CA, USA) per manufacturer’s instruction. cDNA synthesis and target gene amplification were carried out on the CFX96 Real-Time System (Bio-Rad) at 50 °C for 10 min followed by 40 cycles of at 95 °C for 10 sec and 60 °C for 30 sec.
Yang Y., Huycke M.M., Herman T.S, & Wang X. (2016). Glutathione S-transferase Alpha 4 Induction by Activator Protein 1 in Colorectal Cancer. Oncogene, 35(44), 5795-5806.
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2

Quantitative PCR Analysis of RNA Levels

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NucleoSpin RNA II kit (Clontech) was used to purify total RNA. cDNAs were synthesized using High-Capacity cDNA reverse transcription kit (Applied Biosystems). cDNAs analyzed by qPCR utilizing Power SYBR Green and the 7900T Fast Real-Time PCR System (Applied Biosystems). The following primers were used to detect RNA levels of CFTR (1–2), GFP, (3–4), Actin (5–6), NAA10 (7–8), NAA15 (9–10), NAA20 (11–12), and NAA25 (13–14) (see Table 3). qPCR data was analyzed using the comparative CT method [21 (link)].
Vetter A.J., Karamyshev A.L., Patrick A.E., Hudson H, & Thomas P.J. (2016). N-Alpha-Acetyltransferases and Regulation of CFTR Expression. PLoS ONE, 11(5), e0155430.
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3

Isolation and Quantification of PSC-Derived RNA

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On predetermined days, hydrogels containing encapsulated PSCs were collected, placed in DNase/Rnase-free microtubes, and flash frozen using liquid nitrogen. Samples were then stored at −80°C until use. To extract RNA, frozen gels were homogenized in 600 µL of lysis buffer (NucleoSpin RNA II kit, Clontech) and were subjected to two additional cycles of freeze-thaw in order to lyse cells. Lysates were purified using NucleoSpin Filters. Following purification, 600 µL of Rnase-free 70% ethanol was added to the lysates and mixed thoroughly. The mixtures were then transferred to NucleoSpin RNA columns for RNA extraction following manufacturer’s protocol. Using 30 µL of Dnase/Rnase-free water, the isolated RNAs were eluted and then quantified by UV spectrometry (NanoDrop 2000, Thermo Scientific). Using PrimeScript RT reagent kit (Clontech) the total isolated RNA were converted into single-stranded cDNA. Gene expression level was assessed by quantitative real-time PCR and analyzed following a published protocol. Real-time PCR primer sequences can be found listed in Table S1.
Liu H.Y., Greene T., Lin T.Y., Dawes C.S., Korc M, & Lin C.C. (2016). Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells. Acta biomaterialia, 48, 258-269.
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4

RNA Extraction and Reverse Transcription

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Total RNAs were extracted from cultured cells by Nucleospin RNA II kit (Clontech Laboratories, Mountain View, CA, USA) according to manufacturer’s protocol, and subjected to reverse transcription as previously described [49 (link)].
Bernardini M., Brossa A., Chinigò G., Grolez G.P., Trimaglio G., Allart L., Hulot A., Marot G., Genova T., Joshi A., Mattot V., Fromont G., Munaron L., Bussolati B., Prevarskaya N., Fiorio Pla A, & Gkika D. (2019). Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis. Cancers, 11(7), 956.
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5

RNA Isolation and Q-RT-PCR Analysis

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Total RNA was prepared using the RNeasy RNA isolation kit (Qiagen) or NucleoSpin RNA II Kit (Clontech) and cDNA was synthesized by cDNA EcoDry Premix Double Primed (Clontech) kit. Q-RT-PCR was performed with SoFast Evagreen Supermix (Bio-Rad) as per manufacturer's protocol.
Kumar P P., Emechebe U., Smith R., Franklin S., Moore B., Yandell M., Lessnick S.L, & Moon A.M. (2014). Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex. eLife, 3, e02805.
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6

Quantitative gene expression analysis of encapsulated cells

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Samples of cell-laden hydrogels were collected in DNase/RNase-free microtubes, flash-frozen in liquid nitrogen and stored in −80°C. Tota l RNA was isolated from the encapsulated cells with NucleoSpin RNA II kit (Clontech). The concentration and purity of RNA were obtained by NanoDrop 2000 Spectrophotometer (Thermo Scientific). Next, purified RNA samples were converted into complementary DNA (cDNA) with PrimeScript RT Reagent Kit (Clontech, TaKaRa). For Taqman® array experiments, only samples with concentration greater than 100 ng/mL, 260/280 > 2.0, 260/230 > 1.8 were used. cDNA samples were diluted to 100 ng/mL and mixed with same volume of Taqman® fast universal master mix. For 96 well TaqMan® Array Gene Signature Sets, 10 μl mixture was deposited in each well and detected by Applied Biosystems 7500 Fast Real-Time PCR machine. Three biological replicas were used for each experimental condition. Additional qPCR on EMT-related genes was performed using cDNA and SYBR Premix Ex TaqII kit (Clontech) with appropriate primers as listed in Table S1. The relative gene expression levels were analyzed by the 2−ΔΔCT method with GAPDH as the internal control (i.e., housekeeping gene) and the expression level of respective gene in the control group (i.e., Gel/PEG gel with no HA and no stiffening) as the external control.
Liu H.Y., Korc M, & Lin C.C. (2018). Biomimetic and enzyme-responsive dynamic hydrogels for studying cell-matrix interactions in pancreatic ductal adenocarcinoma. Biomaterials, 160, 24-36.
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7

RNA Isolation and qRT-PCR Analysis

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RNA was isolated using the Nucleospin RNA II kit (Clontech Laboratories, Inc., Mountain View, CA, USA). 1μg of RNA was subjected to reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). cDNA was used as template for quantitative RT-PCR using ABsolute Blue QPCR SYBR Green Low ROX Mix (Thermo Scientific) and Applied Biosystems 7500 Fast (Life Technologies, Carlsbad, CA, USA).
Sato M., Rodriguez-Barrueco R., Yu J., Do C., Silva J.M, & Gautier J. (2014). MYC is a critical target of FBXW7. Oncotarget, 6(5), 3292-3305.
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8

Quantitative RT-PCR for mRNA Expression

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Total RNA was extracted from cells using the Nucleo Spin RNA II kit (Clontech, Mountain View, CA) as per the manufacturer's instructions. The quantity and quality of RNA was determined using NanoDrop 1000 (Thermo Fisher Scientific). Complementary DNA prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem, Carlsbad, CA) was mixed with primers and the iQ SYBR Green Premix (Bio‐Rad, Hercules, CA) for quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) to detect the expression of mRNA transcripts. The primer sequences are listed in Supporting Information Table S1. Levels of relative mRNA expression were calculated using the 2−ΔCt method by referencing to those of the internal control ubiquitin.
Wang J.F., Lee M., Tsai T., Leiferman E.M., Trask D.J., Squire M.W, & Li W. (2019). Bone Morphogenetic Protein‐6 Attenuates Type 1 Diabetes Mellitus‐Associated Bone Loss. Stem Cells Translational Medicine, 8(6), 522-534.
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9

Profiling Interferon Signaling in STING KO Neurons

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Primary neurons were isolated and cultured from E14 mice embryos of wild-type (WT) and STING knockout mice using the method as previously described.30 (link) RNA was extracted and quantified as using a NucleoSpin RNA II Kit (Clontech, catalog number—740955-250). cDNA was generated using RT2 First strand kit (Qiagen, catalog number—330401) and RT2 SYBR Green qPCR Mastermix (Qiagen, catalog number—330503). cDNA was used in mouse interferons and receptors 96 wells array plates (Qiagen, catalog number—PAMM-064ZC) for performing RT2 Profiler PCR Array. Raw data were analyzed using Qiagen gene globe web portal (http://www.qiagen.com/geneglobe). GraphPad Prism 9.3 was used to generate the heat map.
Kundu N., Kumar A., Corona C., Chen Y., Seth S., Karuppagounder S.S, & Ratan R.R. (2022). A STING agonist preconditions against ischaemic stroke via an adaptive antiviral Type 1 interferon response. Brain Communications, 4(3), fcac133.
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10

Molecular Profiling of Gastric Stem Cells

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For the Cck2r, Lgr5, Notch1, Dll1, Sox2, Axin2, Ascl2 and Numb mRNA expression analysis from sorted Cck2r-CreERT+ cells and sorted Lgr5-GFP+ cells, the gastric single cells from antrum were isolated as described previously (Hayakawa et al., 2015b (link); van Es et al., 2012 (link)). Single antral stem cells were sorted from Cck2r-CreERT;R26-tdTomato mice at 24 hours after TAM induction, or from Lgr5-DTR-GFP mice. All primer sequences were listed in Table S1.
For the Cck2r, Lgr5, Notch1, Dll1 and Gastrin mRNA expression analysis from gastric antral tisuue, the longitudinal strips of gastric tissue from the anterior wall as well as the posterior wall were harvested and snap-frozen in dry ice and kept in a −80 °C freezer until processed for analysis. Total RNA was extracted with Nucleospin RNA II kit (Clontech) and cDNA was synthesized by Superscript III First-strand Synthesis System for RT-PCR (Thermo Fisher Scientific). Expression levels of indicated genes were quantified by Real-Time PCR (qPCR) assays using SYBR Green and 7300 Real Time PCR System. Primer sequences used in this experiment are available upon request.
Chang W., Wang H., Kim W., Liu Y., Deng H., Liu H., Jiang Z., Niu Z., Sheng W., Nápoles O.C., Sun Y., Xu J., Sepulveda A., Hayakawa Y., Bass A.J, & Wang T.C. (2020). Hormonal suppression of stem cells inhibits symmetric cell division and gastric tumorigenesis. Cell stem cell, 26(5), 739-754.e8.
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