scholarly journals A highly parallel, automated platform enabling individual or sequential ChIP of histone marks and transcription factors

2019 ◽  
Author(s):  
Riccardo Dainese ◽  
Vincent Gardeux ◽  
Gerard Llimos ◽  
Daniel Alpern ◽  
Jia Yuan Jiang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Throughput ◽  
Dynamic Range ◽  
Throughput Capacity ◽  
Specific Gene ◽  
Human Lymphoblastoid Cell ◽  
Sequential Chip ◽  
Automated Platform ◽  
On Chip ◽  
Low Sensitivity

AbstractDespite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry and molecular biology to address the major limitations of standard ChIP-seq. The resulting approach, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2h), has a wide dynamic range (from 106 to 500 cells), is high-throughput (up to 64 parallel, antibody- or sample-multiplexed experiments) and is compatible with both histone mark and transcription factor ChIP. In addition, FloChIP’s interconnected design allows for straightforward chromatin re-immunoprecipitation, thus constituting the first example of a microfluidic sequential ChIP-seq system. Finally, we demonstrate FloChIP’s high-throughput capacity by performing ChIP-seq of the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing novel insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B-cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.

scholarly journals A parallelized, automated platform enabling individual or sequential ChIP of histone marks and transcription factors

2020 ◽  
Vol 117 (24) ◽  
pp. 13828-13838 ◽  
Author(s):  
Riccardo Dainese ◽  
Vincent Gardeux ◽  
Gerard Llimos ◽  
Daniel Alpern ◽  
Jia Yuan Jiang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dynamic Range ◽  
Specific Gene ◽  
Histone Marks ◽  
Human Lymphoblastoid Cell ◽  
Sequential Chip ◽  
Standard Chip ◽  
Automated Platform ◽  
On Chip ◽  
Low Sensitivity

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2 h), has a wide dynamic range (from 106to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP’s interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.

scholarly journals Real-Time Reverse Transcription-PCR Assay for Future Management of ERBB2-based Clinical Applications

1999 ◽  
Vol 45 (8) ◽  
pp. 1148-1156 ◽  
Author(s):  
Ivan Bièche ◽  
Peter Onody ◽  
Ingrid Laurendeau ◽  
Martine Olivi ◽  
Dominique Vidaud ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Real Time ◽  
Gene Amplification ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Mrna Level ◽  
Throughput Capacity ◽  
Specific Gene ◽  
Pcr Assay ◽  
Erbb2 Gene

Abstract Background: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations. Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA. Results: The dynamic range was &gt;1000-fold. The relationship between Ct and log starting concentration was linear (r2 ≥0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (&gt;5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (&lt;5) in breast cancers without gene amplification. Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.

scholarly journals Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

2012 ◽  
Vol 18 (1) ◽  
pp. 116-127 ◽  
Author(s):  
John D. Graef ◽  
Lisa C. Benson ◽  
Serguei S. Sidach ◽  
Haiyang Wei ◽  
Patrick M. Lippiello ◽  
...  
Keyword(s):  
High Throughput ◽  
Dose Response ◽  
Throughput Capacity ◽  
Response Curves ◽  
Uncompetitive Inhibition ◽  
Compound Screening ◽  
Methodological Approaches ◽  
Dose Response Curves ◽  
Automated Platform ◽  
Electrophysiological Methods

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3β4 and hα4β2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC50 values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHβE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (&gt;10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

scholarly journals 1.0 V-0.18 µm CMOS Tunable Low Pass Filters with 73 dB DR for On-Chip Sensing Acquisition Systems

Electronics ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 563
Author(s):  
Jorge Pérez-Bailón ◽  
Belén Calvo ◽  
Nicolás Medrano
Keyword(s):  
Power Consumption ◽  
Dynamic Range ◽  
Low Voltage ◽  
Cutoff Frequency ◽  
Cmos Technology ◽  
Active Area ◽  
Current Steering ◽  
Low Pass ◽  
On Chip ◽  
Low Pass Filters

This paper presents a new approach based on the use of a Current Steering (CS) technique for the design of fully integrated Gm–C Low Pass Filters (LPF) with sub-Hz to kHz tunable cut-off frequencies and an enhanced power-area-dynamic range trade-off. The proposed approach has been experimentally validated by two different first-order single-ended LPFs designed in a 0.18 µm CMOS technology powered by a 1.0 V single supply: a folded-OTA based LPF and a mirrored-OTA based LPF. The first one exhibits a constant power consumption of 180 nW at 100 nA bias current with an active area of 0.00135 mm2 and a tunable cutoff frequency that spans over 4 orders of magnitude (~100 mHz–152 Hz @ CL = 50 pF) preserving dynamic figures greater than 78 dB. The second one exhibits a power consumption of 1.75 µW at 500 nA with an active area of 0.0137 mm2 and a tunable cutoff frequency that spans over 5 orders of magnitude (~80 mHz–~1.2 kHz @ CL = 50 pF) preserving a dynamic range greater than 73 dB. Compared with previously reported filters, this proposal is a competitive solution while satisfying the low-voltage low-power on-chip constraints, becoming a preferable choice for general-purpose reconfigurable front-end sensor interfaces.

scholarly journals Subtype-associated epigenomic landscape and 3D genome structure in bladder cancer

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tejaswi Iyyanki ◽  
Baozhen Zhang ◽  
Qixuan Wang ◽  
Ye Hou ◽  
Qiushi Jin ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Transcription Factor ◽  
Cancer Cell ◽  
Genome Structure ◽  
Transcription Factor Binding ◽  
Specific Gene ◽  
Open Chromatin ◽  
Factor Binding ◽  
3D Genome ◽  
Genome Wide

Abstract Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown. Result We determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. Conclusion In summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.

scholarly journals Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts

2006 ◽  
Vol 12 (1) ◽  
pp. 140-144 ◽  
Author(s):  
Michael K. Hancock ◽  
Myleen N. Medina ◽  
Brendan M. Smith ◽  
Anthony P. Orth
Keyword(s):  
High Throughput ◽  
Dynamic Range ◽  
Cell Lysis ◽  
Single Step ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Activity Measurements ◽  
Simple Detection ◽  
Reporter Assays ◽  
Orbital Mixing

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

scholarly journals Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1019
Author(s):  
Teresa von Linde ◽  
Gzona Bajraktari-Sylejmani ◽  
Walter E. Haefeli ◽  
Jürgen Burhenne ◽  
Johanna Weiss ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Peptide Bond ◽  
Peptide Transporter ◽  
Systemic Absorption ◽  
Screening Assay ◽  
Limit Of Quantification

The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.

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