Total RNA was extracted from hippocampus or hippocampal primary cultures using RNAqueous Micro Kits (Ambion) and reverse transcribed using High Capacity RNA-to-cDNA Kits (Applied Biosystems). qRT-PCR was performed on an ABI 7500 Fast Sequence Detection System (Applied Biosystems). For RNA expression analysis, TaqMan assays (Applied Biosystems) were used with Gapdh as a reference gene (Supplemental Experimental Procedures ). For ChIP-qPCR, EpiTect ChIP qPCR assays (QIAGEN) were used. All reactions were performed with RT2 SYBR Green, following the manufacturer’s recommended cycling conditions, and subjected to melting curve analysis. All changes of gene expression were determined using the 2−ΔΔCT method (Futai et al., 2013 (link); Livak and Schmittgen, 2001 (link)).
Epitect chip qpcr assay
Manufactured by Qiagen
Sourced in United States, Germany
The EpiTect ChIP qPCR Assay is a laboratory equipment product designed for chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analysis. It provides a complete solution for the detection and quantification of specific DNA sequences associated with proteins of interest in a sample.
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Lab products found in correlation
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Epiquik chip kit, by Epigentek (1 mentions)
7300ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Anti h3k4me3, by Epigentek (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Magnify chromatin immunoprecipitation system kit, by Thermo Fisher Scientific (1 mentions)
Ez magna chip a g kit, by Merck Group (1 mentions)
Ultrasonic disruptor ud 201, by TOMY (1 mentions)
Faststart dna master sybr green 1, by Roche (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
5 protocols using epitect chip qpcr assay
1
Quantitative RNA Expression Analysis
2
Chromatin Immunoprecipitation Assay for H3K4me3
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Molecular events in the kidneys of LPS-treated mice were monitored using a chromatin immunoprecipitation (ChIP) assay to immunopurify soluble chromatinized H3K4 me3 sequences using specific antibodies and the EpiQuik ChIP kit (EPIGENTEK, Brooklyn, NY, USA). The antibodies used included normal mouse IgG as the negative control, anti-RNA polymerase II as the positive control, and anti-H3K4 me3 (EPIGENTEK, Brooklyn, NY, USA). The DNA concentration was quantified using a NanoDrop UV spectrometer (NanoDrop Technologies, Wilmington, DE, USA). A SYBR green assay (LightCycler 480 SYBR Green I Master; Roche, Mannheim, Germany) was performed using the 7300HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using primers for mouse cathepsin L (EpiTect ChIP qPCR Assays, QIAGEN).
3
Chromatin Immunoprecipitation (ChIP) Analysis
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Chromatin immunoprecipitation (ChIP) analyses were performed on chromatin extracts according to manufacturer’s specifications of MAGnify Chromatin Immunoprecipitation System kit (Invitrogen). Sheared chromatin was immunoprecipitated with 5 µg of the following antibodies: anti-β-arrestin1 (Clone 10 cat. 610550 BD Biosciences) or normal mouse IgG, provided by the kit, was used as negative control. Eluted DNA was amplified by qPCR using EpiTect ChIP qPCR Assay (Qiagen) for the indicated genes (Mouse Cdc25a, Trp73, Cdkn1b, Casp3, Casp7, Zeb1, Zeb2, Birc5, Vim and Fn1). As control we used Actin and Gapdh gene. Data are presented as input percentage enrichment over background.
4
ChIP Assay for PATZ1 Binding
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Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's protocol (EZ–Magna ChIP A/G kit; Millipore, Billerica, MA). Cells were incubated for 12 h with PIP3 or medium alone before cross-linking protein complexes to DNA using 1% formaldehyde (Sigma-Aldrich). Samples were sonicated on ice (Ultrasonic Disruptor UD-201; TOMY, Tokyo, Japan) to generate 200-1000 DNA fragments. DNA was incubated with Protein A/G magnetic beads alone or with beads plus anti-PATZ1 (H-300; Santa Cruz Biotechnology) or IgG isotype control antibodies. The beads were pelleted with a magnetic separator and purified DNA was analyzed by SYBR green real time PCR using EpiTect ChIP qPCR Assay (Qiagen, Valencia, CA) with specific primers against the proximal region of human pp4r2 gene promoter (GPH1009388(+)01A; +363 bp from the transcription start site). Data are expressed as percentage relative to the untreated control.
5
ChIP-qPCR Assay for NF-κB
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Cells (1.2 × 10 5 cells per well in six-well plates) were treated as described in Section 2.2 and then stimulated for 30 min.
After incubation chromatin was prepared using Chromatrap Enzymatic Shearing Kit (Chromatrap, Wrexham, UK) following manufacturer's instructions and was checked on 1% agarose gel for appropriate size of sheared chromatin. The chromatin was further processed using Abcam high sensitivity ChIP Kit (Abcam, Cambridge, UK) according to manufacturer's instructions and the immunoprecipitation was carried out using rabbit antihuman-NF-𝜅B p65 antibody (Active Motif, La Hulpe, Belgium) and normal rabbit IgG (Cell signaling, Beverly). After purification, DNA was analyzed using Fast Start DNA Master SYBR Green I and the Light Cycler 1.5 device (Roche Diagnostics, Mannheim, Germany). ChIP DNA was normalized according to the input DNA. The sequence of primers and PCR conditions are given in Table S2, Supporting Information. The primers for NF-𝜅B BS present in human IL-8 promoter were purchased from Qiagen (Hilden, Germany) Epitect ChIP qPCR assay.
After incubation chromatin was prepared using Chromatrap Enzymatic Shearing Kit (Chromatrap, Wrexham, UK) following manufacturer's instructions and was checked on 1% agarose gel for appropriate size of sheared chromatin. The chromatin was further processed using Abcam high sensitivity ChIP Kit (Abcam, Cambridge, UK) according to manufacturer's instructions and the immunoprecipitation was carried out using rabbit antihuman-NF-𝜅B p65 antibody (Active Motif, La Hulpe, Belgium) and normal rabbit IgG (Cell signaling, Beverly). After purification, DNA was analyzed using Fast Start DNA Master SYBR Green I and the Light Cycler 1.5 device (Roche Diagnostics, Mannheim, Germany). ChIP DNA was normalized according to the input DNA. The sequence of primers and PCR conditions are given in Table S2, Supporting Information. The primers for NF-𝜅B BS present in human IL-8 promoter were purchased from Qiagen (Hilden, Germany) Epitect ChIP qPCR assay.
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