Oxygen and miRNAs in Cosmetology and Dermatology

A recent review by Nadim et al. casts some light on a Cosmetology and Dermatology, where circulating biomarkers, though studied to a certain extent so far, are yet unknown for many skin models. (1) A first element to have in mind when considering the skin-related experimental model is the “Oxygen level”. Oxygen levels may contribute to different findings, and the in vitro models used so far may not be so physiologically relevant as initially thought.

Hypoxia and miRNAs

Tissue oxygenation (which is a major part of the cell microenvironment) regulates the expression of the microRNAs called oxymiRs. OxymiRs may be categorized into three groups:

  1. microRNAs whose expression is directly modied by the Oxygen partial pressure,
  2. microRNAs whose expression is indirectly (pH, metabolites, etc.) modified by the Oxygen partial pressure, andpO2 percentage levels in various tissues
  3. microRNAs that target mediators of Oxygen sensing pathways to regulate biological networks for cell survival.

Examples of miRNAs important in skin physiology include miR-17, miR-21, miR-24, miR-27, miR-29b, miR-99 family, miR-125, miR-146a, miR-155, miR-203 and miR-205, among many other.

For example, in injured tissue, disruption of the vascular supply is associated with a low oxygen partial pressure, or hypoxia, which induces the expression of specic microRNAs referred to as HypoxamiRs (included in group (1) of the oxymiRs classification).

miR-210, known as the master HypoxamiR, is robustly induced under hypoxic conditions in nearly all kinds of cells. Under hypoxia, miR-21 also induces angiogenesis by targeting PTEN, leading to activation of AKT and ERK1/2 signaling pathways.

Transfection Factors and microRNAs

Other transcription factors such as p53 and NF-kB have been shown to affect the expression of microRNAs under hypoxia/anoxia conditions.

Numerous studies have demonstrated the modulation of microRNA expression and particularly that of the miR-200 family with oxidative stress due to excessive ROS levels.

Up to now, very few studies have shown the role of the intermediate Oxygen level (physioxia) in regulating microRNA expression in skin cells. Taken together, these results strongly support the idea that physioxia should be an important criterion in determining the microRNA expression level and consequently protein expression and skin functions.

Therefore, when trying to understand the role of microRNAs in skin models, it is important to choose a cell culture system that is as physiological as possible, and have the tools to analyse the different biomarkers (no only microRNA, but also signaling pathways, secretome, etc).

Should you like to have more information on this review, or know what we can do for you to support you with cell culture technologies with controlled oxygen levels or to support you for your biomarker discovery studies, do not hesitate to contact me by leaving a message below.

Source:

(1) Nadim M. et al. “Physioxia and MicroRNAs As Key Factors in the Skin Microenvironment” (2015) FSCC Magazine, Vol. 18 – #1, pp: 35-43

miRNAs: potent biomarkers in cancer research?

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Expression signals of validated miRNAs that differentiated pancreato-biliary cancer from non-malignant abnormalities (A), or from cancers of other types (B).

A recent paper by Kojima, M. et al. has found a signature of miRNAs to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.

Namely, a combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively.

In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort.

Finding of this eight miRNAs was possible by using Toray’s 3D profiling technology. This finding is especially important, as it is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology.

microRNAs are stably present in peripheral blood, and are therefore a good candidate for finding prognostic or diagnostic biomarkers.

Studying only the miRNAs available in the literature may limit the novelty of the biomarkers found, so for a wide variety of diseases, a profiling is mandatory in order to find really specific circulating biomarkers.

Toray’s technology is available in Europe as fee-for-services via tebu-bio Laboratories (see Press Release). By exploring the full miRnome (composed by 2,000 miRNAs) with Toray’s 3D-Gene® technology, one might identify slight miRNA expression level changes (including low abundance miRNAs) in blood samples, biopsies FFPE specimens…

Interested in miRNA profiling in cancer research? Leave a message below or browse tebu-bio’s 3D-Gene® miRNA profiling platform web page!

Array profiling to study PPAR-alpha in progenitor cell

Peroxisome proliferator-activated receptor alpha (PPAR-alpha also known as NR1C1)  regulates a myriad of biological processes. It is a key modulator of lipid metabolism.

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services)

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services).

Vergori, L. et al. have shown in murine models how PPAR-alpha regulates endothelial progenitor cell maturation and myeloid lineage differentiation via a NADPH oxidase-dependent mechanism. (1) All the data described in this publication suggest that PPAR-alpha, in murine models, is a critical regulator of recruitment, homing and maturation of Bone Marrow-derived progenitor cells.

This conclusion was made (in part) possible with the analysis of secretome markers by using C-Series profiling arrays for the analysis of bone marrow-derived cells in PPAR-alpha wild-type vs. KO mice.

RayBio® Membrane-Based Antibody Arrays (C-Series) are tools for screening and comparing expression levels of many cytokines between samples. C-series Arrays are available as ready-to-use kits or lab services. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012.

tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Interested in studying the secretome biomarkers relevant in your model for Cardiovascular diseases?

Leave your comment or contact us!tebu-bio: European RaybioTech's Certified Laboratory service provider

Source:
(1) Vergori L. et al. “PPARa regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice” (2015) Stem Cells – 33 (4) :1292-303. DOI: 10.1002/stem.1924.

Phosphorylation studies made with Antibody Arrays

Oropharyngeal squamous cell carcinomas (OSCCs) can be either Human papillomavirus (HPV)-positive or HPV-negative. Profiles of druggable Receptor Tyrosine Kinases (RTKs) are different in both groups, as shown in a paper by Cortelazzi, B. et al. The authors chose a cohort of 17 HPV-positive and 59 HVP-negative Formalin-Fixed OSCCs, in order to study E5 expression and RTK alterations. RTK activation was explored in further 12 Frozen OSCCs.

HPV-positive and HPV-negative OSCCs showed different RTK profiles, including differences in E5 and HER2 levels, as well as in HER3 activation and heterodimerisation (HER3/EGFR, also seen for HER2/EGFR). PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases.

The authors stated that, for HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. They also indicate that in HPV-negative cases, HER3 may be a promising druggable biomarker that would deserve further investigation. Finally, PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCCs, particularly in HPV-positive/PI3KCA-mutated OSCCs.

This study was possible in part thanks to an approach based on arrays to detect multiple biomarkers in biological samples at the same time, followed by validation using simplex technologies. These experimental approaches are known for cytokine profiling but also exist for phosphorylation studies (RTKs, EGFR, mTOR phospho-pathways…).

To ensure top quality in the data obtained, you might outsource your biomarker profiling and validation to certified service providers fully trained. tebu-bio: European RaybioTech's Certified Laboratory service provider

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012 but also by Quansys BioSciences and FullMoon BioSystems technologies. The Biomarkers team will be proud to support you to publish novel discoveries by providing innovative discovery & validation tools.

Normalisation assays in biomarker studies – all’s well that starts well

The normalisation of quantitative assay data is critical when interpreting effective biological system status. With cells grown in culture and lysed, a simple total protein determination such as the Bradford assay (developed by Marion Bradford at the University of Georgia in 1976) can be enough by giving an estimate of the total cellular proteins. However, this type of measurement, along with Lowry and other dye binding assays, can be prone to errirs due to various factors such as detergent, chelators… Standardization also involves the use of a protein of interest. Here again, the protein used is crucial for the accuracy of the overall assay.

[Read more…]

Discovery of new biomarkers… 3 tips regarding controls

One of the recurrent questions that we get at the Biomarkers team at tebu-bio is on what controls should be included in a given experiment. Either if the experiment is done by researchers in their lab, or if we collect their samples and perform the analysis in our lab, a good design starts by using the most convenient controls.

One of the controls is related to the study itself. In this sense, definition of what a control population is, and how we want to study it vs. a cohort of patients has been discussed elsewhere in a proteomics post. Today, we will put our spotlight on the “technical” controls, i.e. those related to the technique itself.

Control # 1 – positive control

Obvious. We need to check that the technology we are using is able to detect what we want to detect. And before starting with unknown samples, we need to check that it works in samples we know well.Multicoloured wells - Blog Thumbnail

Ideally, a positive control should be as similar as the samples we want to analyse. In this sense, samples for a given health state (be it with a disease or not), are commercially available, or they can be found if not yet available. tebu-bio has a network of collaborations with private companies that can provide validated samples, fulfilling all ethical and clinical criteria.

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Background can be an issue…only if you are not able to detect it. If you can see it, then you can either modify your protocol, or discard that sample. Picture shows an example of what can be seen with slightly-hemolysed plasma.

Alternatively, we can use recombinant or chemically synthesised controls. If we take the example of recombinant proteins (either in pure form or spiked in biological samples), there are many which are commercially available, or they can be made upon demand. Here it is important that the recombinant protein is very similar to the one found in an organism, including glycosylations and other post-translational changes. In this sense, for many control proteins, HEK293 is preferred over E.coli as an expression system.

Control #2 – negative control

Water. Or PBS. Or not?

Ideally, a negative sample should be as similar as to our case samples as possible. Meaning that it has the same clinical and biological parameters than our samples of interest…

Commercially available samples mentioned previously can be a good approach. For immunological studies involving cell culture supernatants, it is important to include a control with the culture medium only, as FBS can affect the specificity of the assay and can render false-negatives due to background.

Background due to FBS is not detected by technologies such as ELISAs or bead-based, whereas it is detected in optical-based technologies such as arrays and Q-plex.

Control #3 – technical replicates

Every technology has an inherent coefficient of variation (CV).

Genomic technologies usually are under 5 % CV. Immunoassays are around 10 to 25 % (or even more). This means that, for some biomarkers where the difference between healthy vs. disease is small, CV may hide the relevance of these biomarkers. This is especially dramatic in studies related to signal transduction, where differences are usually very small.

6-plicates in an antibody array.

6-plicates in an antibody array.

A way to make sure about whether a result comes from real biology or artificial CV is the performance of replicates.

Triplicates (or even 4-plicates) have been popular with ELISA users. Nowadays, however, most researchers perform duplicates, and repeat the analysis of the sample if the results are very discordant. This approach is quite practical, and still allows to have accurate results in a sensible way (i.e. not doing 4-plicates for every sample!).

Antibody arrays in the market usually have replicates spotted onto the same slide (from duplicates to 8-plicates), which can be considered as semi-independent technical replicates. Therefore, there is no need, in most cases, to perform additional technical replicates.

In any case, every project is different, so we are continously advising our customers on what is the best approach for one given study. From its design to the technology best suited to get the best results, we are glad to contribute to the advance of the understanding of biomarkers in several diseases.

Wondering what controls to include in your experiment? Don’t hesitate to contact us!

Identification of a MicroRNA signature for Fibromyalgia diagnosis

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Heatmap of PBMCs miRNome from FM patients and controls. The microarray analysis was performed with Toray’s 3D-Gene Human miRNA Oligo chips (v.16.0; current version is v21.0). Patient samples are labeled (FM 1–11) and controls (C 1–10). Color palette is included to indicate signal intensity.

A recent paper by Cerdá-Olmedo, G. et al. unravels the miRNA signature in fibromyalgia (FM). Diagnosis of FM, a chronic musculoskeletal pain syndrome characterised by generalized body pain, hyperalgesia and other functional and emotional comorbidities, is a challenging process hindered by symptom heterogeneity and clinical overlap with other disorders. No objective diagnostic method exists at present.

This study aimed at identifying changes in miRNA expression profiles (miRNome) of FM patients for the development of a quantitative diagnostic method of FM. In addition, knowledge of FM patient miRNomes would lead to a deeper understanding of the etiology and/or symptom severity of this complex disease.

A broad profiling was first performed using Toray’s technology. miRNAs found were validated by qPCR in a later step. The profiling of FM patients PBMCs showed a marked downregulation of hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p, hsa-miR145-5p and hsa-miR-21-5p (4-fold or more).

Globally, 20% of the miRNAs analyzed (233/1212) showed downregulation of at least 2-fold in patients. This might indicate a general de-regulation of the miRNA synthetic pathway in FM. No significant correlations between miRNA inhibition and FM cardinal symptoms could be identified. However, the patient with the lowest score for mental fatigue coincided with the mildest inhibition in four of the five miRNAs associated with the FM-group.

Therefore, the authors propose a signature of five strikingly downregulated miRNAs (hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-miR143-3p and hsa-miR145-5p) to be used as biomarkers of FM. Validation in larger study groups would be required before the results can be transferred to the clinic, as the authors indicate.

Looking for miRNA signatures? Don’t hesitate to leave your comments below!

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!

Circulating Biomarkers in Cardiovascular Disease

Cardiovascular diseases (CVD) are one of the the primary causes of morbidity and mortality worlwide. Therefore, more accurate Blood cardiac biomarkers (CB) are needed for a correct handling of patients, at the diagnostic and prognostic level. CVDs are a myriad of diseases with different molecular mechanisms, so it is important to have CBs specific for each pathology.

Toray's 3D-Gene sensitive and accurate miRNA and mRNA microarray service by tebu-bio

Toray’s 3D-Gene technology enables sensitive and accurate miRNA and mRNA analysis through tebu-bio services

Biomarkers are a significant tool to better identify high-risk individuals, to diagnose disease conditions promptly and accurately, and to efficiently prognose and treat patients with disease. Circulating biomarkers, such as miRNAs, have been used in other fields (e.g. Oncology), but their use in clinical practice for CVD patients is still scarce. [Read more…]

miRNAs and Autoimmune Glomerulonephritis

Correlation of findings in animal models (e.g. mouse) and its validation in human samples lies at the basis of Translational medicine. Nowadays, hypothesis established in mouse models must, at some stage, be validated in a cohort of patients. [Read more…]