scholarly journals An improved method for quantitative ChIP studies of nuclear receptor function

2019 ◽  
Vol 62 (4) ◽  
pp. 169-177
Author(s):  
Ann Louise Hunter ◽  
Natasha Narang ◽  
Matthew Baxter ◽  
David W Ray ◽  
Toryn M Poolman
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Chromatin Immunoprecipitation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Correct Interpretation ◽  
Receptor Function ◽  
Improved Method ◽  
Chip Data ◽  
Accurate Quantification

Chromatin immunoprecipitation (ChIP) is a valuable tool for the endocrine researcher, providing a means to measure the recruitment of hormone-activated nuclear receptors, for example. However, the technique can be challenging to perform and has multiple experimental steps, risking introduction of error at each. The data produced can be challenging to interpret; several different methods are commonly used for normalising data and thus comparing between conditions. Absolute, sensitive quantification of protein-bound DNA is important for correct interpretation of the data. In addition, such quantification can help the investigator in troubleshooting experiments. Here, we outline a ChIP strategy combining droplet digital PCR for accurate quantification with an internal spike-in control for normalisation. This combination strengthens the reliability of ChIP data and allows the operator to optimise their protocol with greater confidence.

scholarly journals Molecular Mechanisms Underlying the Link between Nuclear Receptor Function and Cholesterol Gallstone Formation

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Mary Carmen Vázquez ◽  
Attilio Rigotti ◽  
Silvana Zanlungo
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Molecular Mechanisms ◽  
Cholesterol Metabolism ◽  
Cholesterol Gallstone ◽  
Gallstone Disease ◽  
Gallstone Formation ◽  
Receptor Function ◽  
Prevalent Disease ◽  
Bile Cholesterol

Cholesterol gallstone disease is highly prevalent in western countries, particularly in women and some specific ethnic groups. The formation of water-insoluble cholesterol crystals is due to a misbalance between the three major lipids present in the bile: cholesterol, bile salts, and phospholipids. Many proteins implicated in biliary lipid secretion in the liver are regulated by several transcription factors, including nuclear receptors LXR and FXR. Human and murine genetic, physiological, pathophysiological, and pharmacological evidence is consistent with the relevance of these nuclear receptors in gallstone formation. In addition, there is emerging data that also suggests a role for estrogen receptor ESR1 in abnormal cholesterol metabolism leading to gallstone disease. A better comprehension of the role of nuclear receptor function in gallstone formation may help to design new and more effective therapeutic strategies for this highly prevalent disease condition.

scholarly journals Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

2014 ◽  
Vol 53 (2) ◽  
pp. 699-701 ◽  
Author(s):  
Eva Malatinkova ◽  
Maja Kiselinova ◽  
Pawel Bonczkowski ◽  
Wim Trypsteen ◽  
Peter Messiaen ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Terminal Repeat ◽  
Genomic Dna Isolation ◽  
Plasmid Isolation ◽  
Hiv 1 ◽  
Accurate Quantification

Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

scholarly journals Unorthodox Transcriptional Mechanisms of Lipid-Sensing Nuclear Receptors in Macrophages: Are We Opening a New Chapter?

2020 ◽  
Vol 11 ◽  
Author(s):  
Zsolt Czimmerer ◽  
Laszlo Halasz ◽  
Laszlo Nagy
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Protein Protein Interaction ◽  
Lipid Sensing ◽  
Transcriptional Memory ◽  
Genome Level ◽  
Generation Sequencing

Work over the past 30 years has shown that lipid-activated nuclear receptors form a bridge between metabolism and immunity integrating metabolic and inflammatory signaling in innate immune cells. Ligand-induced direct transcriptional activation and protein-protein interaction-based transrepression were identified as the most common mechanisms of liganded-nuclear receptor-mediated transcriptional regulation. However, the integration of different next-generation sequencing-based methodologies including chromatin immunoprecipitation followed by sequencing and global run-on sequencing allowed to investigate the DNA binding and ligand responsiveness of nuclear receptors at the whole-genome level. Surprisingly, these studies have raised the notion that a major portion of lipid-sensing nuclear receptor cistromes are not necessarily responsive to ligand activation. Although the biological role of the ligand insensitive portion of nuclear receptor cistromes is largely unknown, recent findings indicate that they may play roles in the organization of chromatin structure, in the regulation of transcriptional memory, and the epigenomic modification of responsiveness to other microenvironmental signals in macrophages. In this review, we will provide an overview and discuss recent advances of our understanding of lipid-activated nuclear receptor-mediated non-classical or unorthodox actions in macrophages.

Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR

2016 ◽  
Vol 95 (5) ◽  
pp. 739-744 ◽  
Author(s):  
Miguel Waterhouse ◽  
Marie Follo ◽  
Dietmar Pfeifer ◽  
Nikolas von Bubnoff ◽  
Justus Duyster ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Jak2 V617f Mutation ◽  
Chronic Myeloproliferative Neoplasms ◽  
V617f Mutation ◽  
Accurate Quantification

scholarly journals Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Cristian Koepfli ◽  
Wang Nguitragool ◽  
Natalie E. Hofmann ◽  
Leanne J. Robinson ◽  
Maria Ome-Kaius ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Malaria Parasites ◽  
Human Malaria ◽  
Accurate Quantification

scholarly journals Nuclear Receptor Cofactors in PPARγ-Mediated Adipogenesis and Adipocyte Energy Metabolism

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-11 ◽  
Author(s):  
Emily Powell ◽  
Peter Kuhn ◽  
Wei Xu
Keyword(s):  
Transcriptional Regulation ◽  
Energy Metabolism ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Proper Function ◽  
Peroxisome Proliferator ◽  
Clear Understanding ◽  
Receptor Function ◽  
Peroxisome Proliferator Activated Receptor ◽  
Transcriptional Cofactors

Transcriptional cofactors are integral to the proper function and regulation of nuclear receptors. Members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors are involved in the regulation of lipid and carbohydrate metabolism. They modulate gene transcription in response to a wide variety of ligands, a process that is mediated by transcriptional coactivators and corepressors. The mechanisms by which these cofactors mediate transcriptional regulation of nuclear receptor function are still being elucidated. The rapidly increasing array of cofactors has brought into focus the need for a clear understanding of how these cofactors interact in ligand- and cell-specific manners. This review highlights the differential effects of the assorted cofactors regulating the transcriptional action of PPARγand summarizes the recent advances in understanding the physiological functions of corepressors and coactivators.

scholarly journals Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays

2017 ◽  
Vol 19 (2) ◽  
pp. 236-243 ◽  
Author(s):  
Willem H. Zoutman ◽  
Rogier J. Nell ◽  
Mieke Versluis ◽  
Debby van Steenderen ◽  
Rajshri N. Lalai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pcr Assays ◽  
Accurate Quantification

scholarly journals Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse

2020 ◽  
Vol 169 ◽  
pp. 115213 ◽  
Author(s):  
Michael A. Jahne ◽  
Nichole E. Brinkman ◽  
Scott P. Keely ◽  
Brian D. Zimmerman ◽  
Emily A. Wheaton ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr
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