Genejet rna isolation kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The GeneJet RNA Isolation Kit is a tool designed for the purification of total RNA from various biological samples, including cells, tissues, and microorganisms. The kit utilizes a silica-based membrane technology to capture and purify RNA, allowing for efficient extraction and subsequent analysis or downstream applications.

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24 protocols using genejet rna isolation kit

Hepatic Lipid and Xenobiotic Metabolism

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Expression of key genes regulating hepatic lipid and xenobiotic metabolism were examined by real-time polymerase chain reaction (PCR) as previously described (Mulligan et al., 2017 (link)). Genes governing lipid hepatic lipid metabolism include the lipogenic genes sterol response element binding protein-1c (Srebp-1c), fatty acid synthase (Fas), and acetyl-CoA carboxylase-1 (Acc-1), the fatty acid translocase Cd36, and genes governing fatty acid oxidation including carnitine palmitoyl transferase-1a (Cpt-1a) and peroxisomal acyl-coenzyme A oxidase-1 (Acox-1). Genes governing xenobiotic metabolism include cytochromes 3a11 (Cyp3a11), 1a2 (Cyp1a2), 2b10 (Cyp2b10), and 2c29 (Cyp2c29). These cytochromes are murine orthologues of the prominent human cytochrome P450 enzymes (Nelson et al., 2004 (link)). To measure gene expression, total RNA was isolated (Genejet RNA Isolation kit; Thermo Fisher) then cDNA was transcribed (Verso cDNA Synthesis Kit; Thermo Fisher) from 1 μg of total RNA. cDNA amplification was measured by Sybr Green detection (Sybr Select Master Mix; Thermo Fisher) with beta-actin as housekeeping gene. Alterations in target gene expression were determined by the ΔΔCt method as previously performed (Howell et al., 2009 (link); Mulligan et al., 2017 (link)). Data are expressed as the fold change from the average of standard diet fed vehicle-treated animals.

Howell GE I.I.I., Kondakala S., Holdridge J., Lee J.H, & Ross M.K. (2018). Inhibition of cholinergic and non-cholinergic targets following subacute exposure to chlorpyrifos in normal and high fat fed male C57BL/6J mice. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 118, 821-829.

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Preparation of p53 Variant RNA Templates

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The dsDNA templates for the preparation of P0-Δ40p53, P1-Δ40p53, P1-Δ40p53(L), P1-Δ40p53(S), P1-Δ40p53(Δ57), P1-Δ40p53(ΔHDM2), p53-554 and isolated hairpin G56-C169 RNAs had been obtained previously in our laboratory [38 (link),39 (link),63 (link)]. For transcription in vitro, the dsDNA templates were first linearised with Csp45 or Xba1 restriction enzymes (Thermo Fisher, Walthan, MA, USA) in accordance with the manufacturer’s protocols. Transcription reactions were performed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, Walthan, MA, USA) as recommended by the manufacturer’s protocol and with an addition of 4 mM guanosine. After the transcription, RNAs were purified using the GeneJet RNA Isolation Kit (Thermo Fisher, Walthan, MA, USA).

Janecki D.M., Swiatkowska A., Szpotkowska J., Urbanowicz A., Kabacińska M., Szpotkowski K, & Ciesiołka J. (2021). Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5′-Terminal Region of p53 mRNA. International Journal of Molecular Sciences, 22(24), 13306.

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Quantifying Gene Expression in CNS Tissue

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RNA was isolated from the CNS tissue using a GeneJET RNA isolation kit (Thermo Fisher), and cDNA was synthesized using a M-MLV Reverse Transcriptase (Promega, Madison, WI). qPCR analysis was performed using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA) using TaqMan reagents (Applied Biosystems) or SYBR green master mixes (Applied Biosystems). The gene expression was normalized to the housekeeping gene gapdh.

Kim D., Le H.T., Nguyen Q.T., Kim S., Lee J, & Min B. (2019). IL-27 attenuates autoimmune neuroinflammation via Treg/Lag3-dependent but IL-10-independent mechanisms in vivo. Journal of immunology (Baltimore, Md. : 1950), 202(6), 1680-1685.

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RNA Extraction and RT-qPCR Analysis

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RNA was isolated using the GeneJET RNA isolation kit (Thermo Fisher Scientific #K0732) according to the manufacturer’s instructions. Genomic DNA was digested using an on-column DNase step (Sigma-Aldrich #04716728001) for 15 min. RNA was converted into cDNA using the High Capacity cDNA Reverse Transcriptase kit (Thermo Fisher Scientific #43-688-13) according to the manufacturer’s instructions. cDNA was synthesised according to the following steps: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min.
RT-qPCR was performed by using the Brilliant III SYBR® Green QPCR Master Mix (Agilent #600882) or DyNAmo HS SYBR Green (Thermo Scientific #F410L) and the QuantStudio 3. The following RT-qPCR cycling parameters were used: initial denaturation on 95 °C for 10 min, 40 cycles of 95 °C for 20 s, 57 °C for 30 s and 72 °C for 30 s, finished by a dissociation step 65–95 °C (0.5 °C/s). Samples were run in technical triplicates. Fold change expression was determined using the 2^−ΔΔCT method.

Vringer E., Heilig R., Riley J.S., Black A., Cloix C., Skalka G., Montes-Gómez A.E., Aguado A., Lilla S., Walczak H., Gyrd-Hansen M., Murphy D.J., Huang D.T., Zanivan S, & Tait S.W. (2024). Mitochondrial outer membrane integrity regulates a ubiquitin-dependent and NF-κB-mediated inflammatory response. The EMBO Journal, 43(6), 904-930.

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Quantification of mCx45 Transcripts in MDCK Cells

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MDCK clones expressing mCx45 WT and 6E were seeded and cultured to confluence. Cells were rinsed 2x with 1x PBS and lysed for RNA content using the GeneJet RNA Isolation kit (Thermo) per manufacturer protocol. Equivalent masses of RNA were reverse transcribed using the MuMV-RT kit (NEB) per manufacturer protocol with OligoDT priming. Equal volumes of each RT reaction were used as template for standard PCR reactions with primers to mCx45-WT/6E (5′- ACAGTAAAAGGAGGGAACTTGAT-3′ and 5′- TGGCTTTGTTTTGCTTGTAGG-3′) and GAPDH (5′- GTAGTGAAGCAGGCATCGGA-3′ and 5′- GTCGAAGGTGGAAGAGTGGG-3′) using Taq Green Master Mix (Thermo). Cycling was carried out as follows: Initial denaturation 95°C for 2 min, followed by 30 cycles of 95°C denaturation for 30 sec, 54.5°C annealing for 30 sec, and 72°C extension for 30 sec, followed by final extension of 5 min at 72°C. Equal samples volumes were analyzed by 2% Agarose/TAE electrophoresis spiked with ethidium bromide. No reverse transcriptase reactions were used to subtract genomic background. Quantification was done using NIH ImageJ software, normalizing samples to GAPDH loading control. Quantification was done using 3 independent replicates.

Trease A.J., Capuccino J.M., Contreras J.E., Harris A.L, & Sorgen P.L. (2017). Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization. Journal of molecular and cellular cardiology, 111, 69-80.

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Quantitative analysis of HUNK gene

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RNA was prepared by using the GeneJet RNA isolation kit (Thermo Scientific). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific). The resulting cDNA was used to perform quantitative RealTime PCR (QRT-PCR) using the Bio-Rad myIQ system. PrimePCR SYBR Green Assay for human HUNK was purchased from Bio-Rad. Primers for GAPDH are Forward-TGCACCACCAACTGCTTAGC and Reverse-GGCATGGACTGTGGTCATGAG.

Phelps-Polirer K., Abt M.A., Smith D, & Yeh E.S. (2016). Co-Targeting of JNK and HUNK in Resistant HER2-Positive Breast Cancer. PLoS ONE, 11(4), e0153025.

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Transcriptome Analysis of Inflamed Lungs

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RNA was isolated from inflamed lung tissues or FACS sorted cells using a GeneJet RNA isolation kit (ThermoFisher, Waltham, MA cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI). Real time PCR analysis was performed using an ABI7500 real time PCR system (Applied Biosystem, Waltham, MA), a SYBR master mix (Applied Biosystem), and gene specific primers.

Jang E., Nguyen Q.T., Kim S., Kim D., Nga Le T.H., Keslar K., Dvorina N., Aronica M.A, & Min B. (2017). Lung infiltrating Foxp3+ regulatory T cell cells are quantitatively and qualitatively different during eosinophilic and neutrophilic allergic airway inflammation but essential to control the inflammation. Journal of immunology (Baltimore, Md. : 1950), 199(12), 3943-3951.

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Breast cancer gene expression analysis

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Total RNA was isolated from JIMT-1 cells treated with 250 µM aCT1 or PBS for 24 h using the GeneJet RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was analyzed using the Breast Cancer 360™ Panel, composed of 758 genes relevant to breast cancer biology and 18 housekeeping genes, on the nCounter platform (NanoString Technologies, Seattle, WA, USA) as described by the manufacturer. NanoString nSolver 4.0 software was used for the analysis of gene expression values, which were normalized using the housekeeping genes and log2 transformed. The software incorporates the R statistics program. N = 3 control or aCT1-treated samples per group were analyzed.

Baker K.M., Abt M., Doud E.H., Oblak A.L, & Yeh E.S. (2024). Mapping the Anti-Cancer Activity of α-Connexin Carboxyl-Terminal (aCT1) Peptide in Resistant HER2+ Breast Cancer. Cancers, 16(2), 423.

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Quantitative real-time PCR analysis

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RNA was isolated from livers or cells using the GeneJet RNA isolation kit (Thermo Scientific, Waltham, MA). RNA concentration was measured using the Nanodrop 2000 (Thermo Scientific, Waltham, MA) and 1.5 μg RNA per sample was used for cDNA synthesis using the Verso cDNA kit (Thermo Scientific, Waltham, MA). cDNA was amplified with gene specific primers and detected with the Absolute Blue SYBR Green ROX Mix (Thermo Scientific, Waltham, MA) in the StepOne Plus Realtime PCR machine (Applied Biosystems, Grand Island, NY). 18S RNA was used as an invariant control. The standard curve method of analysis for RNA quantitation was utilized using dilutions of corresponding cDNA plasmids as the standards. The primer sequences for S1P, PCSK9, LDLR, SREBP1c, SREBP2, ACC1, FAS and HMGCoA reductase were as described in a previous publication [6 (link)]. All other primer sequences for real-time PCR were obtained from the Primer Bank (http://pga.mgh.harvard.edu/primerbank/).

Basu D., Huq A., Iqbal J., Hussain M.M., Jiang X.C, & Jin W. (2015). Hepatic S1P deficiency lowers plasma cholesterol levels in apoB-containing lipoproteins when LDLR function is compromised. Nutrition & Metabolism, 12, 35.

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Gene Expression Analysis of Hunk in Mice

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RNA was isolated using the GeneJet RNA isolation kit (Thermo Scientific). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific). RealTime PCR using PrimePCR mouse Hunk (Bio-Rad) was performed using the Bio-Rad myIQ. Hunk mRNA levels were normalized to Gapdh levels. Primers for Gapdh are: Forward-GCACAGTCAAGGCCGAGAAT, Reverse-GCCTTCTCCATGGTGGTGAA

Zambrano J.N., Williams C.J., Williams C.B., Hedgepeth L., Burger P., Dilday T., Eblen S.T., Armeson K., Hill E.G, & Yeh E.S. (2018). Staurosporine, an inhibitor of hormonally up-regulated neu-associated kinase. Oncotarget, 9(89), 35962-35973.

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