High resolution structure of the TRPA1 channel protein

One of the hottest news last month was the high resolution structure of the TRPA1 ion channel protein.

Immunohistochemical staining of TRPA1 on mouse intestine with TRPA1 polyclonal antibody (Cat. nr 157PAB11992)

IHC staining on mouse intestine with TRPA1 polyclonal antibody (Cat. nr 157PAB11992). Source: Abnova | tebu-bio.

TRPA1 is a sensor for environmental noxious agents or signaling molecules produced endogenously. The structure was solved in the presence of agonist and antagonist ligands by using single-particle electron cryo-microscopy. (1)

This smart experimental method, which has revealed several unexpected TRPA1 structural features, shows that structural  information thus discovered might greatly assist Life Researchers in the design of better antagonists which will represent the next generation analgesic and anti-inflammatory agents.

Interestingly, several TRPA1-related agents are now well characterized for further in vitro characterization.

Several useful agents for studying TRPA1 channels are available:

  • TRPA1 antagonist HC-030031 inhibits the ion channel (cat. nr 21910-1065)
  • TRPA1 antagonist A-967079 is a cell permeable TRPA1 blocker  (cat. nr 21910-1055)

    Immunogen part (green) from the 4th cystoplasmic loop of the human TRPA1 used to produce human TRPA1 specific antibody (Cat. nr OSR00130W)

    Human TRPA1 modelization. The synthetic from the 4th cytoplasmic loop of human TRPA1 (highlighted in green) was used as the antigen to produce the highly specific human TRPA1 antibody (Cat. nr 214OSR00130W). Source: Osenses | tebu-bio.

  • TRPA1 antagonist AP-18 is a selective TRPA1 channel blocker (cat. nr 21910-1127)
  • TRPA1 agonist Polygodial displays analgesic activity via desensitization of sensory neurons (cat. nr 21910-1145)

 

Thanks to our friends from Focus Biomolecules for this post !

rRNA depletion, poly(A) enrichment, or exonuclease treatment?

RNASeq studies are hampered by the pervasive excess of reads mapping to ribosomal RNA (rRNA), notorious for greatly reducing the amount of useful mRNA sequencing data. Here we highlight the advantages and disadvantages of the various approaches have been developed to address this problem.

Source: DOI: 10.1371/journal.pone.0096094

Source: DOI: 10.1371/journal.pone.0096094

 

1) rRNA depletion with magnetic beads: Technologies such as Ribo-zero™, Ribominus™, and MICROBExpress™ seem to be based on similar technology that uses rRNA depletion probes in combination with magnetic beads to deplete rRNAs from a sample, thus achieving the goal of enriching the representation of mRNAs. Based on our interactions with end-users, Ribo-zero™ customers have been quite pleased with this technology, although some have found it a bit pricey. As a result, some scientists have adapted home-made protocols either using the Organism-Specific Probe Selection software or by designing probes empirically based on the reads they see in their samples. In both cases users custom order Biotin-TEG DNA oligonucleotides in combination with BioMag® Nuclease-Free Streptavidin Particles to complete their own protocols.

2) Poly(A) enrichment: Scientists working in eurkaryotes find that simply purifying poly(A) mRNA with BioMag® Oligo (dT)20 Particles is the most cost-effective approach. The BioMag® SelectaPure mRNA System is a complete low cost kit for isolating mRNA. Clearly users wishing to study histone mRNAs or those of prokaryotes will have to find another solution.

3) rRNA depletion by nuclease: Some of the most straightforward approaches for rRNA depletion involve simple digestion of rRNA with Terminator™ 5′-Phosphate Dependent Exonuclease. While a mature mRNA will carry an m7GTP cap on its 5′ end protecting it from the 5′-to-3′ exonuclease activity, rRNAs and tRNAs carrying only a 5′ phosphate will be degraded. A number of published reports have used this exonuclease approach which seems to be particularly interesting when users have complex environmental samples with multiple prokaryotic species. Alternative strategies involve the use of DNA probes complementary to the rRNA prior to digestion with RNAse H, which degrades only the RNA strand in an RNA:DNA hybrid. RNase-Free DNase I is then used to eliminate the DNA Probes. A report in Nature Methods (DOI:10.1038/nmeth.2483) comparing multiple approaches for RNASeq library prep determined that the Hybridase™ Thermostable RNase H-based approach was superior for low quality, degraded RNA samples.

4) aRNA amplification: Very common in microarray protocols such as those using Illumina BeadChip, the linear amplification of antisense RNA (aRNA) also called cRNA seems like an interesting approach. Kits like the TargetAmp™TargetAmp™-Nano, and TargetAmp™-Pico aRNA Amplification Kits are designed to amplify the input poly(A) mRNA from very low input (down to 10pg) RNA samples such as those obtained by laser capture microdissected tissue or by flow sorting. After amplification, sufficient sample is present for microarray analyses of gene expression. Based on technology developed by James Eberwine for single cell analysis, the TargetAmp kits have been used in RNASeq protocols with very low quantity samples (For example see: Matsumura et al. and Li et al.).

TargetAmp

5) Rolling Circle Amplification: Similar to the Eberwine-based methods, Rolling Circle amplification protocols involve first the reverse transcription of mRNA with the goal of amplifying the cDNA, primarily for single-cell applications (see Pan et al.). These protocols are quickly gaining popularity and make use of CircLigase to circularize full-length single-stranded cDNA or T4 DNA Ligase kits such as Fast-Link™ DNA Ligation Kits for double-stranded cDNA circularization. Use of Phi29 DNA polymerase is then possible, despite the fact that Phi29 normally requires templates of at least 3-4kb. While it is clear to see how reverse transcription using oligo(dT) will enrich mRNA reads relative to those mapping to tRNA and rRNA, such an enrichment would not be possible in prokaryotes. The rolling circle amplification protocol has been adapted to transcriptome analysis in single prokaryotic cells by making use of random hexamers combined with Terminator™ 5′-Phosphate Dependent Exonuclease described in #3 above (see Kang et al.). Interestingly, the protocol makes use of thiophosphate-linked RNA random hexamers to reduce the formation of primer-dimers and to protect against Phi29 DNA polymerase exonuclease activity.

Diagram illustrating rolling circle DNA replication (credit: credit Madeleine Price Ball)

Diagram illustrating rolling circle DNA replication (credit: Madeleine Price Ball)

Need help building your protocol for enrichment of mRNA reads? Contact the technical specialists at tebu-bio!

Legal notes: TargetAmp™ and Ribo-zero™ are trademarks of Epicentre-Illumina. Ribominus™ and MicrobeExpress™ are trademarks of Life technologies.

Wouldn’t you like to spend less money on your reagents?

We all have certain consumer buying habits. But we also know that sometimes we do not necessarily get the best products at the most price-attractive conditions, if we simply buy the same products from the same source again and again…

What is true for our private behavior, most probably applies to our habits when it comes to buying research reagents for our laboratory. [Read more…]

Good news for Illumina mRNA array users

Recently, Illumina informed their customers that their whole-genome arrays for mouse and FFPE-human samples were to be discontinued. This came as a shock to some researchers that had already started studies using them, or had grants approved and would use them in the future.

Considering other technologies when you have one that is working is really difficult. In Science as well as in life in general, if something works, don’t touch it. That said, if the products you are using are discontinued, you really have to start looking for alternatives.dna5_img_01

Today, let’s take a look at an alternative to Illumina microarrays… that in fact has some advantages, including lower background and higher robustness, among other benefits.

This alternative is based on the 3D-Gene technology developed by Toray. We have discussed this technology, and its use for miRNA studies, in previous posts, but today we will focus on its use for mRNA studies.

The mRNA Oligo chip from Toray was selected from the well-established oligo DNA set for microarray from Operon Biotechnologies Inc. – AROSTM v 3.0 and v 4.0. The selection was made with a focus on genes with substantial annotation information. Therefore, a researcher can perform an analysis without being misled by ambiguous information.
The main public databases used in the design of the probes include Ensembl human database, NCBI human RefSeq and H-Invitational Database.

tebu-bio, Toray Reach Agreement for miRNA, mRNA Profiling TechnologyArrays are available for human (25k), mouse (24k) and rat (20k). Access to this technology is easy, as it is also provided as a service by tebu-bio’s laboratorios located near Paris. You only have to tell them when to collect your samples, and they’ll take care of the whole process!

If you’re interested in learning more about these arrays, as well as a comparison with other microarrays in the market, leave your questions below!

Flow cytometry intracellular staining

Intracellular Flow Cytometry enables the identification and analysis of signaling and functional markers within cells from samples containing heterogeneous cell populations. Many cell types can be thus identified through their intracellular Flow Cytometry patterns and key intracellular signaling pathways and their respective post-translational modifications can be conveniently analysed. Behind these unique performances, Intracellular Flow Cytometry require anyhow optimized immunoreagents for cell permeabilization, intracellular staining and fluorescent detection. [Read more…]

Differentiate between K48- and K63-specific Ubiquitination

Ubiquitination is a common post translational protein modification that occurs through an isopeptide linkage between the C-terminus of ubiquitin and the ε-amino group of a lysine residue on the target substrate. Ubiquitin itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in further ubiquitination, generating poly-Ub chains. The ability of Ubiquitin to form polymers through various lysines as well as its NH2-terminus appears to be central to the versatility of this system in regulating a variety of cellular processes. [Read more…]

D-dimer and Cardiovascular Disease

Fibrinogen is the main protein of the blood coagulation system. It consists of two identical subunits that contain three polypeptide chains: alpha, beta and gamma. The process of blood coagulation results in the activation of fibrinogen into fibrin by thrombin and fibrin polymerization. Fibrin clot is then digested by plasmin, and fibrin degradation products of different molecular weights are released into the bloodstream.

D-dimer is one of these fibrinogen degradation products, and is a biomarker for Cardiovascular damage. Therefore, it is widely used in many detection systems in the market, including ELISAs and lateral flow tests.

Clone 8D3, which has been used in many of these detection systems, is nolonger available. So unless you have the hybridoma in your facility, if you were using 8D3, you will have to consider switching to another monoclonal. [Read more…]

Live cell imaging in action: Killer T-cells hunt their target

In a recent video posted on “Cambridge University’s YouTube channel”, Professor Gillian Griffiths and co-workers prove how the Life Science Community can benefit from modern and innovative cell imaging technologies for understanding cellular activity.

In their recent study (1) and film (2), the research team captured real-time behaviour of Cytotoxic T lymphocytes (CTLs) hunting down, moving and killing cancer cells.

This study clearly illustrates the major breakthrough related to new high spatial and temporal resolution, multi-colour, 3D time-lapse imaging microscopy techniques, which image the entire cell volume and display cellular events at micrometre level.

Watch this fascinating video:

Killer T-Cell Credit: Gillian Griffiths/Jonny Settle . 

Sources:

Professor Gillian Griffiths, Dr Yukako Asano and Akex Ritter – Cambridge Institute for Medical Research – Department of Medicine of the Clinical School (NIH OxCam programme with funding from the Wellcome Trust)

1) Ritter A.T. et al. “Actin depletion initiates events leading to granule secretion at the immunological synapse” (2015) Immunity 42, 864–876. DOI: 10.1016/j.immuni.2015.04.013

2) Body’s ‘serial killers’ captured on film destroying cancer cells – See more at: http://www.cam.ac.uk/research/news

Imaging techniques compatible with Alvetex 3D culture

As scientists better understand the benefits of growing cells in three dimensions (3D) and routinely adopt 3D culture techniques, methods for visualising cells must also be adapted and optimised.

HaCaT AlvetexThe most common and routinely used technique for tracking two dimensional (2D) cell cultures is light microscopy. Traditional 2D monolayer cultures are highly transparent and within a single optical plane. The minimal light diffraction and diffusion presented by the plastic surface allows the collection of focussed microscopic images. Cells cultured in genuine 3D environments, such as in Alvetex®Scaffold, present some of the same constraints as tissue samples or biopsies, in that simple, live observation of cultures via phase microscopy is not optimal.

There are however, other techniques that can be implemented which will allow the user to monitor culture progress easily and effectively in 3D. [Read more…]

5 Tips for Flow Cytometry

Flow cytometry, even if somehow a “classical” technique, is still very valuable in Immunology. Uses of flow cytometry vary from cell identification & sorting based on membrane molecules, characterisation and quantification of secreted and intracellular molecules, as well as, more recently, analysis related to exosomes.

Following our series of technical tips for common experiments in Life Sciences (phospho-WB, secondaries, IF, ELISA, primary cell culture), we present today a series of tips to improve your flow cytometry results. [Read more…]