EXO-DNAc-PS: Isolation of circulating and EV-associated DNA

Circulating DNA is emerging as a novel non-invasive tool for patient’s stratification and disease monitoring.

While most of the research has focused on circulating cell-free (cfDNA) or circulating-tumorcell-(CTC)-derived DNA, extracellular vesicle-(EVs)-associated DNA (EV-DNA) is emerging as a third valuable “liquid biopsy” platform.

Genomic single or double-stranded DNA and mitocondrial DNA have been detected in extracellular vesicles. In particular the majority of double-stranded DNA in extracellular vesicles seems to be associated with tumor derived exosomes (Thakur BK et al. 2014; Kalhert et al. 2014 ) where it represents the entire genome of the cancerous tumor from which exosomes were derived.

This discovery indicates the potential of exosomes as a possible diagnostic tool. Exosomes can easily be obtained from a simple blood sample, so combined with an equally simple method of isolation, they may provide an important diagnostic method in the future.


Get genomic DNA from exosomes with the EXO-DNAc Kit.

This kit combines the ability of our DNA-Prep reagent to co-isolate EVs and circulating DNA from biofluids or culture supernatants with a user friendly system for DNA purification. Isolated vesicles are simply lysed with the appropriate lysis buffer and DNA is purified by spin columns and optimised buffers with a fast turnaround time (approximatively 30 minutes).

Then finally, EXO-DNAc provides a DNA concentrator for concentrating the yield (4 fold concentration) and increasing the purity of the DNA to the levels required for digital PCR analysis.

Click here for further information on the Exo-DNAc Kits 

WHITE PAPER: Increasing Ligation Efficiency and Discovery of miRNAs for Small RNA NGS Sequencing Library Prep with Plant Samples.

MicroRNAs (miRNAs) are 18-22 nucleotide long non-coding small RNAs that regulate protein expression and are involved in various cellular process such as development, growth, and physiology. Many studies have shown that miRNA expression in plants is altered by stress-response and environmental changes. Thus, the analysis of miRNA profiles using next generation sequencing can be beneficial to our understanding of stress tolerance of crops and other plants.

Although miRNAs in plants and animals share many similarities, most mature miRNAs in plants contain 2’-O-methylation at the 3’ end. Typically, ligation of a 3’ adapter via the 3’ OH of the miRNA molecule is the first step in small RNA next-generation sequencing (NGS) sequencing library construction. 2’-O-methylation of plant miRNAs reduces ligation efficiency of the 3’ OH, making plant miRNA libraries difficult to generate.

Here, we show that miRNA profiling from bean, wheat, corn, and rice using total RNA inputs can be successfully achieved using the NEXTflex® Small RNA Sequencing Kit v3, without library purification from PAGE gels, and that extending the ligation incubation step increases library yield. Importantly, many putative novel miRNAs were discovered with the NEXTflex® Small RNA Sequencing Kit v3 that were not discovered using other methods. Thus, this protocol allows generation of reduced-bias small RNA libraries from plant total RNA without the tedious step of gel-based size selection, enabling researchers to discover and profile more small RNAs from more samples than with traditional methods.

 

The full white paper is available here:
https://www.newmarketscientific.com/datasheets/Increasing-Ligation-Efficiency-and-Discovery-of-miRNAs-for-Small-RNA-NGS-Sequencing-Library-Prep-with-Plant-Samples.pdf

 

References:
1. Trends in Plant Science, Vol. 17, Issue 4, p196–203, J Cell Physiol. 2016 Feb;231(2):303-13, Curr Opin Plant Biol. 2016 Dec;34:68-76
2. RNA Biology 9:10, 1218–1223; October 2012, The Plant Cell, Vol. 25: 2383–2399, July 2013 3. Nucleic Acids Res, 2011. 39(21): p. e141., RNA, 2011. 17(12): p. 225662., Silence, 2012.
3(1): p. 4., Genome Biol, 2013. 14(10): p. R109.
4. BMC Bioinformatics. 2014; 15(1): 275.

Anti-Human (Cell Membrane Bound) HSP70 Monoclonal

HSP70 is a highly conserved protein that is ubiquitously expressed. It can be found in chloroplasts, eukaryotic cytosol, endoplasmic reticulum, and mitochondria, but also embedded in the cell membrane and in the extracellular space (1,2,3,4). Even though HSP70 is one of the most studied heat shock proteins, the export mechanism and method of membrane insertion are not fully understood. Most proteins in the heat shock family lack a consensus signal for secretion via the ER-Golgi pathway (5).

Researchers have found that HSP70 may be released from cells via exosomes or secretory vesicles (6). Although HSP70 is ubiquitously expressed, there is not much information about its presence on cell surface.

The finding that HSP70 is localized on the cell surface of cancer cells but not normal cells suggests a conformational change of HSP70 in the lower pH environment characteristic of cancer cells (7). The presence of cell membrane embedded HSP70 has been found to increase the stability of cancer cells, thereby protecting tumors from environmental stress (8, 9).

Anti-HSP70 antibody, clone 1H11 (Catalog No. SMC-249) is unique from other commercially available antibodies in that it can bind to the extracellular region of the cell membrane embedded HSP70 protein, allowing researchers to differentiate between membrane bound and intracellular HSP70 across cancer cells types.

Further details are available here:
https://www.newmarketscientific.com/products?utf8=%E2%9C%93&simpleq=SMC-249

References:
1. Gribaldo, S. et al. (1999) J. Bacteriol. 181, 434-443. 2. Sharma, D. & Masison, D.C. (2009) Protein Pept. Lett. 16, 571-581. 3. Sharma, D. et al. (2009) PLoS. ONE.4, e6644. 4. Kampinga, H.H. & Craig, E.A. (2010) Nat. Rev. Mol. Cell Biol. 11, 579-592. 5. Nickel, W. & Seedorf, M. (2008) Annu. Rev. Cell Dev. Biol. 24, 287-308. 6. Multhoff, G. & Hightower, L.E. (1996) Cell Stress. Chaperones. 1, 167-176. 7. De Maio, A. (2011) Cell Stress. Chaperones. 16, 235-249. 8. Horvath, I. & Vigh, L. (2010) Nature. 463, 436-438. 9. Horvath, I., Multhoff, G., Sonnleitner, A., & Vigh, L. (2008) Biochim. Biophys. Acta 1778, 1653-1664.

Glucagon-like Protein Receptor (GLP2R) Antibody

https://www.newmarketscientific.com/GLP2R

An effective anti-glucagon like peptide-2 receptor (GLP2R) antibody (Cat No: 24200).

GLP-2 is an emerging neurotransmitter involved in feeding control in the hypothalamus and brain stem, and has also been shown to regulate satiety through the endocrine system. Additionally, GLP2R agonists have been effective in the treatment of digestive diseases as well as diabetes.

To further understand GLP-2 activity, use of ImmunoStar's GLP2R antibody allows for highly specific staining of neurons in the hypothalamus, brain stem, pancreatic α-cells, enteroendocrine cells in the intestine, as well as enteric neurons, and vagal sensory neurons.

The rabbit antibody for Glucagon-like Protein Receptor is generated for acetyl 65-88 amide sequence targeting rat and human proteins, but not mouse.

Produced by Dr. Mark Brownfield, the peptide sequence encoding the rat GLP2R was retrieved from the NCBI protein database and evaluated using GeneRunner software to generate antigen candidates for antibody production. 

This antibody demonstrates significant labeling of enteroendocrine cells in the intestinal epithelium, as well as cell bodies of vagal afferents in nodose ganglia of the parasympathetic nervous system.

Immunolabelling of Western blot reveals a band of approximately 66 kDa in human and rat tissue.

Further product details are available here:

https://www.newmarketscientific.com/GLP2R

Anti-Human Cell Membrane Bound HSP70 Monoclonal

Small RNA Next Generation Sequencing without the bias

Selected Report:
https://www.newmarketscientific.com/datasheets/Randomized%20Adapters%20for%20Reducing%20Bias%20in%20Small%20RNA-Seq%20LibrariesV2.pdf

Small RNA NGS without the bias

SUMMARY

The past decade has seen an explosion of interest in the small RNA repertoires of animal and plant species, and in understanding the biological function of all types of small RNAs.

Next generation sequencing (NGS) is the most practical method for large-scale small RNA studies that aim to identify and enumerate small RNAs as with qPCR-based approaches, imperfectly matched small RNAs may still be able to hybridise to PCR primers. The same is also true for immobilised probes in microarrays. However NGS for small RNA analysis is also not without its challenges.

Small RNA libraries prepared for sRNA-Seq have been found to contain biases, resulting in libraries that inaccurately represent relative levels of the different small RNAs present in the starting RNA sample. This bias is caused by the T4-phage RNA ligases used during the ligation steps of small RNA library preparation. Ideally, the RNA ligases would show no preference for attaching adapters to small RNAs of different sequences, but in reality this is simply not the case.

A novel solution to overcome ligation bias in sRNA-seq libraries has been developed by Bioo Scientific (Austin TX USA), using a pool of adapters with randomised sequences at the ligation site, where each adapter sequence is present in vast molar excess over any given small RNA in the sample.

To demonstrate the reduced bias when using a randomised adapter strategy, small RNA sequencing libraries were prepared in triplicate from an equimolar mixture of 23 synthetic miRNAs, using either standard or randomised adapters in the ligation steps. Further details and the results are discussed in the link in this blog post.

Figure 1. Standard small RNA sequencing compared to NEXT flex Small RNA Sequencing Kit with randomised adapters.
Libraries were prepared from an equimolar mix of 23 synthetic miRNAs. Each slice in the pie graph represents one miRNA.

https://www.newmarketscientific.com/datasheets/Randomized%20Adapters%20for%20Reducing%20Bias%20in%20Small%20RNA-Seq%20LibrariesV2.pdf

Anti-ERK1/2 Best antibody validated by Antibody Resource

Selected Report:
http://www.stressmarq.com/wp-content/uploads/ERK1-Antibody-Cat-No.-SPC-120-Comparison-Report-Antibody-Resource.pdf

Stressmarq:Anti-ERK1/2 antibody validated by Antibody Resource

SUMMARY

Anti-ERK1/2 polyclonal antibody (Catalog No. SPC-120, referenced as P132 in the report) was independently validated by Antibody Resource for use in western blot on human breast adenocarcinoma (MCF7 cell line) whole cell lysate and mouse embryonic fibroblast (NIH3T3 cell line) whole cell lysate at a dilution of 1:5000, with bands detected at the expected molecular weight of 38-43 kDa. Can also be used in Immunohistochemistry, Immunocytochemistry/Immunofluorescence and flow cytometry.

Immunohistochemistry analysis using Rabbit Anti-ERK1 Polyclonal Antibody (SPC-120). Tissue: backskin. Species: Mouse. Fixation: Bouin’s Fixative Solution. Primary Antibody: Rabbit Anti-ERK1 Polyclonal Antibody (SPC-120) at 1:100 for 1 hour at RT. Secondary Antibody: FITC Goat Anti-Rabbit (green) at 1:50 for 1 hour at RT. Localization: Cytoplasm.

Immunohistochemistry analysis using Rabbit Anti-ERK1 Polyclonal Antibody (SPC-120). Tissue: Inflamed colon. Species: Mouse. Fixation: Formalin. Primary Antibody: Rabbit Anti-ERK1 Polyclonal Antibody (SPC-120) at 1:25000 for 12 hours at 4°C. Secondary Antibody: Biotin Goat Anti-Rabbit at 1:2000 for 1 hour at RT. Counterstain: Methyl Green at 200uL for 2 min at RT.

https://www.newmarketscientific.com/stressmarq

Tips and tricks for antibody production

https://www.newmarketscientific.com/agrisera

Antibody production and the validation process - How to obtain good results

POSTER: The three crucial components for successful antibody production: Antigen, Host and Testing

Contact us for a copy of this poster

https://www.newmarketscientific.com/contact

Highlighting NGS Disease panels

https://www.newmarketscientific.com/ngs

Illumina compatible disease panels

Amplicon sequencing is based on ultra-deep sequencing of PCR products for analysing genetic variations and differences in gene expression. This approach allows researchers to focus interrogation on key regions of genomic interest using a simple workflow.

Amplicon panels allow researchers to sequence large numbers of targeted gene regions from selected samples for analysis of mutational hot spots within a subset of genes or detection of copy number variations (CNVs), well-defined gene fusions, SNPs or Indels. RNA panels are also possible to look at differences in RNA levels of genes of interest.

Using amplicon panels is an easily scalable, simple to use, fast, cost-effective approach which can be applied to a broad range of organisms and/or genes.

The NEXTflex Amplicon Panels include primers flanking the regions of interest, library prep reagents, and barcodes needed for construction of libraries compatible with Illumina sequencing platforms.

Illumina compatible disease panel sequencing kits

BRCA1 and BRCA2 amplicon panels (FFPE & non FFPE) 

Cardiovascular Disease amplicon panel

CEBPA amplicon panel

Colon-1 amplicon Panel

Colorectal Cancer-2 amplicon Panel

Congenital Adrenal Hyperplasia amplicon Panel

Congenital Hyperinsulinism amplicon Panel

Cystic Fibrosis amplicon panel

Diabetes-3 amplicon panel

Diabetes-4 amplicon panel

DMD (Duchenne Muscular Dystrophy) amplicon panel

Epilepsy-2 amplicon panel

HBOC-3 amplicon panel

Marfan's Disease amplicon panel

Mediterranean Fever amplicon panel (fresh or frozen) 

MODY-1 amplicon panel

MODY-2 amplicon Panel

Nephrotic Syndrome-1 amplicon panel

Neurofibromatosis amplicon panel

Obesity-1 amplicon Panel

Obesity-2 amplicon Panel

Periodic Fever-1 amplicon panel

Periodic Fever-2 amplicon panel

Phenylketonuria amplicon panel

TP53 amplicon panel

TP53 amplicon Panel for FFPE

https://www.newmarketscientific.com/ngs

Nature Genetics: Sequencing of the genus Arabidopsis

Selected Paper:

http://www.nature.com/ng/journal/v48/n9/full/ng.3617.html

Sequencing of the genus Arabidopsis identifies a complex history of nonbifurcating speciation and abundant trans-specific polymorphism

Abstract

The notion of species as reproductively isolated units related through a bifurcating tree implies that gene trees should generally agree with the species tree and that sister taxa should not share polymorphisms unless they diverged recently and should be equally closely related to outgroups. It is now possible to evaluate this model systematically. We sequenced multiple individuals from 27 described taxa representing the entire Arabidopsis genus. Cluster analysis identified seven groups, corresponding to described species that capture the structure of the genus. However, at the level of gene trees, only the separation of Arabidopsis thaliana from the remaining species was universally supported, and, overall, the amount of shared polymorphism demonstrated that reproductive isolation was considerably more recent than the estimated divergence times. We uncovered multiple cases of past gene flow that contradict a bifurcating species tree. Finally, we showed that the pattern of divergence differs between gene ontologies, suggesting a role for selection.

https://www.newmarketscientific.com/plantantibodies