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Genome-wide DNA methylation profiling using Infinium assay

Genome-wide DNA methylation profiling utilizing Infinium® assay.

 

OBJECTIVE
Bisulfite sequence evaluation of particular person CpG websites inside genomic DNA is a robust method for methylation evaluation within the genome.
The main limitation of bisulfite-based strategies is parallelization. Each array and next-generation sequencing know-how are succesful of addressing this bottleneck. On this report, we describe the applying of Infinium® genotyping know-how to investigate bisulfite-converted DNA to concurrently question the methylation state of over 27,000 CpG websites from promoters of consensus coding sequences (CCDS) genes.
METHODS
We tailored the Infinium genotyping assay to readout an array of over 27,000 pairs of CpG methylation-specific question probes complementary to bisulfite-converted DNA. Two probes have been designed to every CpG website: a ‘methylated’ and an ‘unmethylated’ question probe.
The probe design assumed that every one underlying CpG websites have been ‘in section’ with the queried CpG website as a consequence of their shut proximity. Bisulfite conversion was carried out with a modified model of the Zymo EZ DNA Methylation™ package.
RESULTS
We utilized this know-how to measuring methylation ranges throughout a panel of 14 totally different human tissues, 4 Coriell cell strains and 6 most cancers cell strains.
We noticed that CpG websites inside CpG islands (CGIs) have been largely unmethylated throughout all tissues (~80% websites unmethylated, β < 0.2), whereas CpG websites in non-CGIs have been reasonably to extremely methylated (solely ~12% websites unmethylated, β < 0.2). Inside CGIs, solely roughly 3-6% of the loci have been extremely methylated; in distinction, exterior of CGIs roughly 25-40% of loci have been extremely methylated. Furthermore, tissue-specific methylation (variation in methylation throughout tissues) was far more prevalent in non-CGIs than inside CGIs.
CONCLUSIONS
Our outcomes display a genome-wide scalable array-based methylation readout platform that’s each extremely reproducible and quantitative. Within the close to future, this platform ought to allow the evaluation of lots of of 1000’s to tens of millions of CpG websites per pattern.
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abx098962-12ml 12 ml
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EpiQuik General Protein-DNA Binding Assay Kit (Colorimetric)

P-2004 96 Assays
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Description: kits suitable for this type of research

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SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent exercise in vitro and in vivo.

FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) sufferers, 2 lessons of FLT3 activating mutations have been recognized: inside tandem duplication (ITD) mutations within the juxtamembrane area (25%-30% of sufferers) and level mutations within the kinase area activation loop (7%-8% of sufferers).
FLT3-ITD mutations are probably the most frequent molecular defect recognized in AML and have been proven to be an unbiased prognostic issue for decreased survival. FLT3-ITD is due to this fact a beautiful molecular goal for remedy.
SU11248 is a lately described selective inhibitor with selectivity for cut up kinase area RTKs, together with platelet-derived development issue receptors, vascular endothelial development issue receptors, and KIT.
We present that SU11248 additionally has potent exercise in opposition to wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. As well as, SU11248 inhibits FLT3-induced VEGF manufacturing. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD fashions: a subcutaneous tumor xenograft mannequin and a bone marrow engraftment mannequin. We present that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors within the subcutaneous tumor xenograft mannequin and prolongs survival within the bone marrow engraftment mannequin.
Pharmacokinetic and pharmacodynamic evaluation in subcutaneous tumors confirmed {that a} single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for as much as 16 hours following a single dose. These outcomes counsel that additional exploration of SU11248 exercise in AML sufferers is warranted.

Age-associated traits of murine hematopoietic stem cells.

Little is understood of age-associated useful modifications in hematopoietic stem cells (HSCs). We studied growing old HSCs on the clonal stage by isolating CD34(-/low)c-Equipment(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) cells from the bone marrow of C57BL/6 mice. A inhabitants of CD34(-)KSL cells progressively expanded as age elevated.
No matter age, these cells shaped in vitro colonies with stem cell issue and interleukin (IL)-Three however not with IL-Three alone. They didn’t kind day 12 colony-forming unit (CFU)-S, indicating that they’re primitive cells with myeloid differentiation potential.
An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells elevated twofold from 2 to 18 mo of age inside a inhabitants of CD34(-)KSL cells in addition to amongst unseparated bone marrow cells.
As well as, we detected one other compartment of repopulating cells, which differed from HSCs, amongst CD34(-)KSL cells of 18-mo-old mice. These repopulating cells confirmed much less differentiation potential towards lymphoid cells however retained self-renewal potential, as steered by secondary transplantation.
We suggest that HSCs progressively accumulate with age, accompanied by cells with much less lymphoid differentiation potential, because of repeated self-renewal of HSCs.

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1176-3
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CaspGLOW Red Active Caspase 8 Staining Kit

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CaspGLOW? Red Active Caspase-8 Staining Kit

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CaspGLOW? Red Active Caspase-8 Staining Kit

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CaspGLOW? Red Active Caspase-9 Staining Kit

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13504 100 tests
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Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*

22797 100 Tests
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20-abx249463
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