1. Addex Pharmaceuticals has developed novel assays to improve GPCR and non-GPCR target screening for allosteric modulator discovery.
2. Their cAMP Phoenyx biosensor provides a dynamic, real-time measurement of receptor activation without altering homeostasis.
3. For GPCRs, the ADX Tags Series 1 assay monitors receptor activation by tagging receptors and binding partners to detect interactions.
4. For non-GPCRs, the Accessory Protein Relocalization Assay detects receptor activation by visualizing accessory protein recruitment.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Recombinant Fab fragments specific for the pfHRPII and pfHSP72 : implications for malaria diagnosis - Parole de junior de la 7e édition du Cours international « Atelier Paludisme » - Ravaoarisoa Elisabeth - Madagascar - elisa@pasteur.mg
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
Recombinant Fab fragments specific for the pfHRPII and pfHSP72 : implications for malaria diagnosis - Parole de junior de la 7e édition du Cours international « Atelier Paludisme » - Ravaoarisoa Elisabeth - Madagascar - elisa@pasteur.mg
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
SuperScript IV Reverse Transcriptase for RNA Analysis | ESHG 2015 Poster PS14...Thermo Fisher Scientific
Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reverse transcriptase and are performing reactions with increasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification.
Back Rapid lead compounds discovery through high-throughput screeningrita martin
High-throughput screening process are used by today most of the drug discovery industries, this process helps pharmaceutical researches to make drug discovery process faster and also increase the quality and quantity of drugs production. This process in combination with robotics, data processing and control software, liquid handling devices and sensitive detectors allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tests
High throughput screening (HTS) at iNovaciaiNovacia AB
iNovacia offers high quality high-throughput screening services supported by a compound collection and screening libraries of highest international standards. iNovacia has a broad experience with all major target families and assay types.
The high throughput screening is the first step of the docking or computational method of drug discovery. This slide will help you to understand the basic things in HTVS.
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
Introduction to the phenomenon of Biased agonism with few examples of receptors exhibiting this phenomenon and an example of drug developed on the basis of biased agonism.
Deepak Pandey, PG Pharmacology, VMMC
BME 302 Cellular Engineering Common Cell & Molecu.docxhartrobert670
BME 302
Cellular Engineering
Common Cell &
Molecular Biology
Assays
1
Common Cell & Molecular
Biology Assays
u Why ?
time=0
signal
Time=t1
?
Qualitatively vs. Quantitatively
What is the
specific
effect of the
trigger e.g.
CELL
PHENOTYPE?
How did this
happen e.g.
Mechanism –
specific cell
transduction
pathway?
Chemical
(GFs)
Mechanical
(pressure)
Common Cell & Molecular
Biology Assays
u Why ?
3
Signals:
Gene expression
CELL
PHENOTYPE
Mechanism:
Specific
signaling
pathway
Common Cell & Molecular
Biology Assays
u How do you characterize the CELL PHENOTYPE?
u Quantification of gene expression through mRNA
quantification
u PCR (end-point and real-time)
u Quantification of proteins (cell epitopes/markers,
secreted cytokines)
u In situ (tissue sections / cells) – Immunostaining
u Microscopy for 2D vs. 3D imaging
u In solutions (supernatants / tissue lysates) –
ELISA
u On the cell surface – FACS
4
Different stages
Ex. Bronchial Epithelial Cell
Differentiation
5
Immunostaining
ELISA
Histochemistry
Basal cells
Epithelial cells
Ex. Long-term self-renewal of
human pluripotent stem cells on
human recombinant laminin511
6
Nature Biotechnology
Volume:
28,
Pages:
611–615
Year published:
(2010)
Nature Biotechnology 28, 611-615 (2010)
FACS
Immunostaining End-point PCR
Real-time PCR
Western Blot
To replace MG
M
G
LN
Gene Transcription & Protein Translation
DNA –> RNA -> Protein
7
Secreted Protein
Internal Protein
PCR
Polymerase Chain Reaction
u Why PCR ?
u Produces many DNA sequence copies without the need of
host cloning (genetic engineering lecture)
u Amplifies known DNA sequences from mRNA reverse
transcription for analysis of gene expression
u Extract mRNA -> rt in cDNA
8
PCR
Polymerase Chain Reaction
9
Cycle 3 Cycle 1
Produces 2 molecules Produces 4
molecules
Produces 8
molecules
Cycle 2
DNA containing
target sequence
to be amplified
Target
sequence
Target
sequence
Target
sequence Template
DNA
primer
DNA
primer
Template
New
DNA
New
DNA
These 2
molecules
match
target DNA
sequence
DNA
primers
DNA
primers
PCR
Polymerase Chain Reaction
u What do you need ?
10
https://www.youtube.com/watch?
v=2KoLnIwoZKU
Traditional end-point PCR
11
Agarose
gel
Buffer
solution
Gel box
Well in gel for
placing DNA
sample
PCR
products
already
loaded to
wells
+
–
+
–
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Micropipettor
adding marker
DNA fragments
to well
Traditional end-point PCR
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Lane with
marker DNA
fragments
Quantification of end-point
PCR
13
Limitations of End-Point PCR
u Poor Precision
u Low sens ...
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
EUGM 2014 - Ádám Andor Kelemen (Hungarian Academy of Sciences): Physicochemic...ChemAxon
Our proposed goal was the design of a physicochemical property-based scoring method for fragment-based drug discovery. The method was developed for sorting commercially available and virtual libraries, suitable for compilation of an aminerg-GPCR targeted fragment library. In our study we examined the physichochemical characteristics of GPCR-promiscous fragments, and concluded the essential parameters into – so called – Desirability Functions. The validation of the score was carried out using the fragment-screening data of an existing fragment-GPCR library. Subsequent goal is an design and establishment of an aminerg-GPCR targeted fragment-library, using the Fragment-GPCR-Score, followed by in-vitro fragment-screening campaign on several GPCR-targets.
6. ADX Tags Series 1.1 Plasma membrane C Activation A) Tag GPCR N BP B) Tag binding partners of activated receptor signal ! N BP BP
7. ADX Tags Series 1.2 Plasma membrane C Activation A) Tag GPCR B) Tag binding partners of inactive receptor BP BP BP NO signal! N N signal ! signal ! N
18. Negative Allosteric Modulation endogenous ligand cell membrane outside cell inside cell active site negative allosteric modulator (NAM) AM binding site COOH
19. Positive Allosteric Modulation endogenous ligand positive allosteric modulator (PAM) active site cell membrane outside cell inside cell COOH
20. Silent Allosteric Modulation endogenous ligand cell membrane outside cell inside cell active site silent allosteric modulator (SAM) COOH
21.
22. FBBA setup: Plasma membrane Fluorescently-Tagged Receptor Time Receptor Fluorescence N SNAP FLUORESCENCE 1 st addition: « Cold » ADX cpd A) 2 nd addition: Tagged ADX cpd B) B) A)