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5 things to understand to land a cloud architect job | killexams.com existent Questions and Pass4sure dumps

Cloud computing is fitting a key manner for organizations to deploy current applications, which is impulsively altering the...

IT job market. And require for cloud architects is notably excessive.

definitely, roughly 11,a hundred cloud architect jobs are presently listed on career web page indeed.com, with salaries starting from $seventy five,000 to more than $one hundred fifty,000 annually. but earlier than touchdown that dream cloud architect job, you exigency to wow competencies employers each and every through the interview system.

listed here are 5 key questions that you may are expecting an agency to inquire of throughout a cloud architect interview, together with tips for the way to respond.

1.      How attain a cloud architect's responsibilities vary from these of different statistics core gurus?

A cloud architect focuses greater on the meta, or huge-photo, view of the statistics focus and fewer on an individual server's configuration and throughput. for instance, cloud architects determine how a company's material authentication system ensures that only licensed personnel entry device resources. by using comparison, a paraphernalia analyst is tasked with tying the authentication paraphernalia to a specific software, reminiscent of Salesforce.com.

2.       where attain you view expertise in three hundred and sixty five days? How about three years?

in Place of rep caught up within the every day grind of facts core and cloud operations, cloud architects must consider ahead. They deserve to breathe blue-sky, huge-photograph thinkers. Cloud architects assess how emerging technologies, fondness biometrics and the web of issues, will influence industry systems and cloud infrastructure. They besides should craft a roadmap that suggests the Place the enterprise' programs are these days and the Place they deserve to breathe in a number of years.

three.       How attain containers healthy into an organization's cloud architecture?

groups are perpetually trying to shape software more portable, and containers are the latest model on that theme -- which makes them a essential technology for cloud architects to understand. First, cloud architects ought to withhold in wit the capabilities containers present. Containers drudgery at a layer above the OS and virtualization utility. Theoretically, they present more portability, however they pay a value for that handy movement: lowered protection. The utility running in the containers doesn't consist of the inherent protection tests discovered at the OS or virtualization layer. consequently, operating containers within a company's records middle and in the back of its security perimeter makes sense, while putting the utility onto a public cloud is a bit of risky. 

4.       What regular interfaces should a company use?

OpenStack has emerged as a key platform, enabling corporations to tie diverse cloud functions together. organizations essentially use free, open supply software as an IaaS platform. OpenStack, which is obtainable below an Apache license, incorporates a group of interrelated components that ply pools of processing, storage and networking supplies.

5.       What cloud architect certifications attain you gain got, or are pursuing?

Cloud architect certification courses Come from two different sources. unbiased training and certification organizations, fondness Arcitura training, CompTIA and EXIN, present supplier-impartial certifications. furthermore, the business's largest providers, such as EMC, Hewlett-Packard, IBM and Microsoft, gain cloud architect certifications geared towards their specific items.

Cloud architects are becoming more everyday. by using realizing solutions to key questions, IT execs can Place themselves to land a high-paying cloud architect job.


All Greek banks viewed passing ECB's stress examine, NBG says | killexams.com existent Questions and Pass4sure dumps

by way of George Georgiopoulos

ATHENS (Reuters) - Greek banks will current the eu valuable bank's (ECB) stress test of their monetary fitness, the manager govt of the nation's 2d-greatest lender, countrywide bank (NBG) <NGBr.AT>, mentioned on Monday.

The ECB will submit the effects of a stress verify of Greece's four greatest lenders - Piraeus <BOPr.AT>, NBG, Eurobank <EURBr.AT> and Alpha <ACBr.AT> - in can besides to permit time for any capital shortfall to breathe filled earlier than Greece leaves its bailout programme in August.

"All Greek banks will stir the stress checks. The outcomes should breathe encouraging," Leonidas Fragiadakis told reporters on the sidelines of a signing ceremony with the eu funding Fund on mortgage amenities for small organizations.

The Greek stress check, aimed at uncovering any capital scarcity earlier than Athens exits its 86 billion-euro (£seventy four.four billion) bailout, can breathe performed separately from an everyday stress verify of other euro zone banks.

look at various outcomes for 33 lenders from different euro zone nations could breathe posted on Nov. 2.

Greek banks were recapitalised thrice due to the fact a debt crossroad exploded in 2010, but are nonetheless stressed by way of 96 billion euros of soured debt. they've dedicated to pursuits to in the reduction of that to sixty five billion euros by using 2019.

"The Greek banking system will emerge more than adequate," Fragiadakis referred to.

The CEO besides observed NBG become looking to approach two chinese buyers over the sale of a majority stake in its insurance unit national coverage, after a blueprint to promote it to Netherlands-based mostly EXIN fell via.

"we are discussing the details of the process to approach both chinese language traders so that the sale takes location as quickly as viable and with a conducive gain an impact on on countrywide bank," Fragiadakis stated.

ultimate month, NBG terminated a deal to promote a seventy five % stake in its coverage subsidiary to EXIN economic functions keeping.

EXIN companions and U.S. Calamos Investments had agreed to purchase the stake for 718 million euros, but the deal turned acrid after a prison row erupted between the two consumers.

(Reporting via George Georgiopoulos; modifying through symptom Potter)


Pearson VUE Cert Survey Sheds pleasing light on IT panorama | killexams.com existent Questions and Pass4sure dumps

Written by Ed Tittel posted: 18 March 2016

When a huge examine facilitator fondness Pearson VUE reviews on certification verify-takers, ambulance it’s probably value taking word. Of particular pastime: who is taking what sorts of certification assessments?

Man with  grave expression using laptopbefore I launch right into a discussion of the responses to a query that Pearson VUE raised on a recent survey performed amongst over 400,000 americans who took a Pearson VUE examination in 2015, there are a brace of things I exigency to examine.

First, while Pearson VUE does administer certification checks for a huge number of certification software sponsors — together with the three 800-pound gorillas of IT certification: Microsoft, Cisco, and CompTIA — it does not administer certification exams for each and every such sponsors and their credentials.

My most amenable ante is that they’ve obtained between 70 and 80 percent of the measure market covered, even though, which makes its effects noteworthy and nigh adequate to representative of the complete certification world to communicate eloquently about what that world is fondness and what’s happening in it.

second, while checking out is a crucial element of earning most certifications, for as a minimum some of them — including essentially the most senior and prestigious credentials — there’s more to getting licensed than passing one or more required assessments. This comprises things fondness documenting drudgery history, obtaining referrals, writing reviews or design files or proposals, providing proof of tutorial historical past and attainment, etc and so on.

Third, i myself gain worked for Pearson education (a sister company to Pearson View) over the years, and gain written books and articles for them (considering the mid-Nineties), blogged for them for five years, and continue to drudgery with and for them on a number of certification-related tasks. I cheerfully confess I could not breathe the most unbiased observer of or stated on their actions, even though I always attain my surest to breathe purpose and correct.

in regards to the Survey

The survey below discussion is called The 2015 cost of Certification Survey (PDF structure). It changed into conducted online from Aug. 3 to Sept. 13, 2015, and worried contacting 400,000-plus individuals who had taken a Pearson VUE examination over the customary 12-month period.

Pearson VUE got 26,603 responses from check-takers each and every over the world, and used these responses to tabulate the consequences and generate the afore-stated file. Pearson describes the ambitions driving the survey as follows:

● To breathe trained greater about examine habits, causes for taking practising and obtaining a certification, paying for behaviors and intentions for future practising and/or trying out.● To capitalize insight into the cost of IT certification for candidates.

The industry provided a small incentive to contributors, in that the primary one hundred individuals in each of three geographic areas acquired a $10 (USD or local equivalent) reward certificates from Amazon for featuring their responses. these areas had been: The Americas, Europe, focus East, and Africa (EMEA), and Asia-Pacific.

usual, the gross survey is pretty wonderful and informative, and intensely a mighty deal charge skimming over, if not reading in comprehensive element. If this sort of issue pursuits you it’s truly value downloading and analyzing.

concerning the question and Its solutions

With each and every this preliminaries and disclaimers in the back of us, recall a glance at the image covered with this weblog publish. The question it raises and whose answers it graphs out reads “in the past twelve months, gain you ever taken a certification examination for any of the following classes? please select each and every that apply.”

Ed Tittel Pearson VUE Survey graphic

The photo goes on to depict a histogram for the properly 10 responses in alphabetic order by sponsor name. I’ve reordered the proper ten programs with the aid of level of participation here:

1. Microsoft: 36.33 p.c 2. Cisco: 23.61 percent three. VMware: eleven.fifty four percent four. HP: 10.29 p.c 5. CompTIA: eight.fifty four % 6. Oracle: 8.15 percent 7. IBM: 2.sixty eight p.c 8. LPI: 2.04 p.c 9. EMC: 1.eighty four p.c 10. EXIN: 1.fifty two percent

First, some observations. while I’m not in any respect shocked to eye two of the three 800-pound gorillas recall first and 2nd Place here (Microsoft and Cisco), I’m more than a bit stunned to view CompTIA in fifth location, in the back of both VMware and HP.

2nd, the overall of each and every contributors who spoke back to this question that fell in the precise 10 changed into 106.fifty four %. (I don’t gain entry to the number of respondents who did recall a certification examination that 12 months at the moment, nor attain i know what number of of the respondents took a brace of certification exam within the equal period. I’ve contacted Pearson VUE to eye if i will rep these numbers).

It is obvious that some respondents took a brace of certification exam, some from numerous exam sponsors (or it wouldn’t gain produced a complete participation cost of greater than 100 percent).

other cert sponsors that registered on this radar included (in alphabetical order): Adobe, Alcatel Lucent, Apple, Appsense, Arista, ASQ, Atlassian, Autodesk, Avaloq, BCS, Brocade, Citrix, CIW, Dassault, Dell, EC Council, Ericsson, fb, Filemaker, Google, IIBA, IIST, ISACA, (ISC)2, ISQI, ISTQB, Juniper Networks, Lenovo, LinkeIn, Nokia, Palo Alto, PeopleCert, PMI, Puppet Labs, QAI, Ruijie Networks, SAP, SAS, Siemens, SQE, Symantec, Teradata, and Tibco.

by means of my count, this makes for a complete of fifty four groups usual. a lot of these gain accustomed and -diagnosed global certification courses – for instance, Adobe, Apple, Autodesk, Brocade, Citrix, EC Council, ISACA and (ISC)2, to identify just a number of – while many extra serve greater confined geographical markets or technology niches.

It does issue to seize an interest cross portion of the total certification panorama, however, and shows how huge and assorted IT authorities’ certficiation hobbies really are.

I’m quiet chewing on the weight of VMware and HP upstaging CompTIA. thus far, I believe that VMware’s Place at No. 3 indicates simply how significant virtualization is to simultaneous IT operations and, with the aid of extension, how vital the cloud has become as smartly.

HP’s presence in fourth locality indicates that this every so often underappreciated technology enterprise has some market heft, in addition to enough accomplice drawing vigor (certification for companion organizations’ technical cadaver of workers is a common hallmark for a lot of companies’ cert classes, including HP’s).

It can breathe pleasing to view if the atomize up into HP industry (commercial laptop methods, application an tech services) and HP Inc. (personal computers and printers) produces some equivalent splits in numbers for the subsequent Pearson VUE survey. I’m guessing probably not, as a result of many of the HP certifications cover products and structures that are actually a share of HPE.

For me the best righteous shock within the suitable 10 changed into EXIN, a corporation that presents certifications in the ITIL and company methods and governance areas. I wager that makes it a pleasing shock, really, as a result of while those disciplines are significant to conducting formal and suitable IT operations, many corporations and agencies had been gradual to comprehend them.

Even showing up on the backside of the reform 10 seems to argue this stuff is ultimately on the radar, and having fun with expanding uptake. respectable information!

There’s lots of unique tips to breathe gleaned from this document, so I’ll doubtless recrudesce to it over again or two. in the meantime, recall a eye for yourself, and spot what you feel.

ed-tittel120in regards to the writer

Ed Tittel is a 30-plus-12 months laptop industry veteran who's labored as a utility developer, technical marketer, consultant, writer, and researcher. author of many books and articles, most advantageous called developer of "exam Cram" IT cert prep bespeak collection. Ed blogs on certification themes for TechTarget, Tom’s IT seasoned, and PearsonITCertification.com, otherwise you can recall a eye at his web page at www.edtittel.com.


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Clearing EX0-113 checks become for each and every intents and purpose unrealistic for the capitalize of me. The test factors were honestly extreme for me to realize. However they illuminated my drawback. I illuminated the 90 inquiries out of a hundred Questions effectively. By essentially referring to the examine manual in brain sell off, I used to breathe prepared to peer the topics properly. besides the top notch exam simulator fondness killexams.Com EX0-113 With achievement cleared this test. I present gratitude killexams.Com for serving the wonderful administrations. Much favored.


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TMap NEXT® Testing Clouds: First Cloud e-book to breathe launched by Sogeti | killexams.com existent questions and Pass4sure dumps

ti, kesä 21, 2011 08:30 CET

A current publication in Sogeti’s TMap® series; TMap NEXT® Testing Clouds is launched.

21st June, Paris/Helsinki: Sogeti, a leading provider of professional technology services, has launched a current publication in its TMap® series, at IBM Innovate 2011 in Orlando USA. TMap NEXT® Testing Clouds is a comprehensive overview of how testing clouds can bring value for the early adopter. The author, Ewald Roodenrijs, describes the cloud industry model for testing, the evolution from Information Technology (IT) to the concept of industry Technology (BT), and the steps that exigency to breathe taken in implementing cloud projects.

Cloud computing is currently regarded as one of the top three IT technologies for the forward thinking CIO. Although the cloud is quiet at an early stage of development, they are starting to view stout growth in cloud-based computing, which is outstripping even the most optimistic predictions. It is increasingly pellucid that the cloud model will supplement, if not entirely replace, mainframe and client/server installations in the years to come. This is based on a compelling value proposition: hasten to market, agility to bring forward or retire service, and the haphazard to stir expenditure from Capital Expenditure (CapEx) to Operational Expenditure (OpEx).

As cloud service adoption becomes ever more wide-ranging, a current global infrastructure is being created that creates significant current opportunities for application character assurance and testing. This current bespeak TMap NEXT® Testing Clouds describes in particular two aspects of the cloud: the industry model and the cloud platform, both of which must breathe tested. This consists of several aspects; testing the infrastructure, cloud-enabled applications, and the skill to gain instant deployable test infrastructure.

Ewald Roodenrijs comments: “This each and every has an impact on the way they attain testing in the future. Testing applications on the cloud is the same as testing applications on a traditional infrastructure. Only what is tested is different”.

TMap NEXT® Testing Clouds is a progress from its TMap® predecessors. It outlines how structured testing, using TMap®, can breathe leveraged within a cloud environment, it is not a step-by-step handbook, but reviews in detail the framework and weight of testing on the cloud, testing cloud strategy (in, on or within the cloud) and the risks involved for companies considering migrating applications onto the cloud, such as security, data integrity, privacy issues, data recovery and performance.

It besides can besides breathe read as a companion or follow-on publication to Seize the Cloud – A Manager’s lead to Success with Cloud Computing[1], exploring in more detail the testing aspects of the cloud.

TMap NEXT® Testing Clouds is available as an e-book in both epub and pdf format and can breathe downloaded for free from the TMap testing website or the Sogeti.com website. Sogeti is increasingly producing e-books as an efficient means of timely distribution as well as enabling the company to reduce its carbon footprint. A printed version is available by using Printing on require (POD) via online bookstores or www.tmapbooks.com.

According to Nijs Blokland, Global Testing Community Lead and Head of Sogeti NL R&D; “Most of us are awake of the benefits of cloud computing, but gain less understanding of the practicalities of successfully and securely implementing cloud applications and using the cloud as a cost-effective source of infrastructure and test tools. This bespeak TMap NEXT® Testing Clouds provides early insight and guidance on these issues, and is further evidence of Sogeti’s innovative thinking in bringing the advantages of both cloud and structured testing together”.

Since launching their Software Testing as a Service (STaaS) globally in 2008, Sogeti has been pioneering the use of cloud-based solutions. As a direct progress of this, Sogeti Netherlands will breathe launching their first Testing Cloud Solution, a comprehensive portal which will provide a complete menu of services, direct access to speedily automated pricing and the skill to upload testing artifacts and assets for cloud-based execution.

For the Dutch company’s clients, this is much more than a front-end website. This portal will provide complete clarity of the service, together with a fixed turnaround time and charge for each of the 22 services, including the creation of a Master Test Plan, Model Based Testing, tool selection, Performance testing, Test Automation, Web Accessibility and Security Testing.

Notes to Editors

About the Author

Ewald Roodenrijs, author of TMap NEXT Testing Clouds, is the Global Lead of the Cloud Testing services within the Sogeti Group and a member of the testing R&D team of Sogeti Netherlands. He is besides the co-author of the bespeak TMap NEXT® – BDTM, contributor to Seize the Cloud, a regular contributor to national and international technical/expert publications, a speaker at numerous international conferences, including most recently at IBM Innovate June 2011, a keen blogger on the Cloud and testing and enthusiastic trainer. He is currently working together with IBM Rational to further develop this cloud offering and recently received the Capgemini-Sogeti Testing Services Innovation Award (Individual) 2011 for his drudgery on Cloud Testing.

About the Book

The bespeak can breathe ordered at major online books store, including Amazon, or directly from the publisher via www.tmapbooks.com or via www.sogeti.com. A printed copy can breathe ordered at www.tmapbooks.com.

Introduction, Chapter 1: An introduction to TMap NEXT® Testing Clouds and Chapter 2 Framework and weight of testing; even in the cloud; of interest to each and every readers.

Chapter 3: The industry in permeate of IT; the Cloud and Chapter 4 Whatever as a Service; cloud-enabled Software Testing as a Service of interest to test managers, program managers and industry managers and IT department managers.

Chapter 5 ‘Testing Cloud Strategy; a stir to 3D’’, Chapter 6 ‘Testing the Cloud; in, on or with...’ and Chapter 7 ‘Cloud risks; worth testing for…’ are mainly enthralling for the target groups of test managers, test coordinators, infrastructure testers and testers.

About the Contributors

The following contributors helped in the creation of the content of this bespeak from Sogeti, Capgemini and besides IBM Rational: Andréas Prins, symptom Buenen, Nick Lloyd, Ramanathan Iyer, Alfonso López de Arenosa, Rob Baarda, Richard Ammerlaan, Erik Smit, Flavien Bouche, Kanchan Apt, Michiel Boreel, Pierre Bedard, Michiel Rigterink, Karl Snider, John Bloedjes, Dan Hannigan, Dirkjan Kaper, plus additional support from Leo van der Aalst, Nicolas Claudon and Clare Argent.

For more information please contact:

Ewald Roodenrijs, Global Lead for Cloud Testing, SogetiTel: +31 (0)6 52 32 77 73Email: ewald.roodenrijs@sogeti.nl

Therese Sinter, Group Corporate Communications Director, Sogeti Tel: +46 70-361 46 21 Email: therese.sinter@sogeti.se

About Sogeti

Sogeti is a leading provider of professional technology services, specializing in Application Management, Infrastructure Management, High‐Tech Engineering and Testing. Working closely with its clients, Sogeti enables them to leverage technological innovation and achieve maximum results. Sogeti brings together more than 20,000 professionals in 15 countries and is present in over 100 locations in Europe, the US and India. Sogeti is a wholly‐owned subsidiary of Cap Gemini S.A., listed on the Paris Stock Exchange.

Together, Sogeti and Capgemini gain developed innovative, business-driven character assurance (QA) and testing services, combining best-in-breed testing methodologies (TMap® and TPI®) and the global delivery model, Rightshore®, to aid organizations achieve their testing and QA goals. Sogeti and Capgemini gain created one of the largest dedicated testing practices in the world, with over 8,200 test professionals and a further 12,500 application specialists, notably through a common focus of excellence with testing specialists developed in India.

In Finland Sogeti is focused solely on providing testing services on the local market.

For more information please visit www.sogeti.com and www.sogeti.fi.

[1] Written by Erik van Ommeren, Martin van den Berg, Sogeti and published by Sogeti March 2010. Seize the Cloud is a lead through the industry and enterprise architecture aspects of Cloud Computing, including 11 cases in which leading organizations share insights gained from their sustain with Cloud.

Yritysesittely Sogeti Finland OySogeti on erikoistunut korkealuokkaisten IT-palvelujen toimittamiseen Suomen markkinoille. Keskitämme Suomessa palvelumme ohjelmistojen testaukseen ja laadunvarmistukseen. Sogeti-konsernin pääkonttori on Pariisissa Ranskassa. Konsernilla on palveluksessaan yhteensä noin 20 000 konsulttia 15 maassa: Belgia, Espanja, Hollanti, Intia, Irlanti, Iso-Britannia, Luxemburg, Norja, Ranska, Saksa, Suomi, Sveitsi, Ruotsi, Tanska ja Yhdysvallat. Pohjoismaissa konsultteja on Suomessa, Ruotsissa, Tanskassa ja Norjassa yhteensä yli 1 100. Sogeti-konsernin yhtiöt ovat Pariisin pörssissä listatun Cap Gemini S.A.:n kokonaan omistamia tytäryhtiöitä.

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Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing | killexams.com existent questions and Pass4sure dumps

Introduction

Exosomes are nanosized membrane vesicles secreted by multiple cell types (Raposo & Stoorvogel, 2013). The term “exosomes” was introduced in 1987 by Johnstone et al. (1987) to depict the “trash” vesicles released by differentiating reticulocytes to dispose unwanted membrane proteins which are known to diminish during maturation of reticulocytes to erythrocytes. Later exosomes gain been implicated in more tangled functions. Raposo et al. (1996) gain demonstrated that exosomes derived from B lymphocytes activate T lymphocytes, suggesting a role for exosomes in antigen presentation in vivo. In early studies, secreted microvesicles were named based on their cellular origins, e.g., archaeosomes, argosomes, dexosomes, epididymosomes, prostasomes, oncosomes, etc. (Raposo & Stoorvogel, 2013). More lately it became obvious that vesicles released by the same cell nature are heterogeneous and can breathe classified into at least three classes based on their mode of biogenesis: (1) exosomes (30–130 nm in diameter), which originate from multivesicular endosome (MVE); (2) microvesicles (100–1000 nm in diameter), which are shed from the plasma membrane; (3) apoptotic bodies (1–4 µm in diameter), which are released from fragmented apoptotic cells during late stages of cell death (Raposo & Stoorvogel, 2013). Various purification procedures including sequential centrifugation protocols gain been proposed to sunder these vesicles for further analysis. Biochemical and proteomic analyses showed that exosomes accommodate specific protein set reflecting their intracellular site of formation. Exosomes from different cell types accommodate endosome-associated proteins, e.g., tetraspanins (CD9, CD63, CD81, CD82), annexins, Rab GTPases and flotillin. Exosomes are besides enriched in proteins involved in MVE formation (Tsg101 and Alix), chaperones (Hsc73 and Hsc90) and cytoskeletal proteins (Raimondo et al., 2011). Furthermore, exosomes accommodate proteins that are specific to the cells from which they are derived (Choi et al., 2012; Sandvig & Llorente, 2012).

The research in the exosome realm has exploded rapidly after the discovery that these microvesicles transport a big number of mRNA and miRNA (Valadi et al., 2007) and that exosomal mRNAs could breathe translated into proteins by recipient cells (Ratajczak et al., 2006; Valadi et al., 2007) and exosomal miRNAs are able to modulate gene expression in recipient cells (Mittelbrunn et al., 2011). Interestingly, inescapable mRNAs and miRNAs were identified as highly enriched in exosomes compared to that of the host cells indicating the being of selective sorting mechanism controlling incorporation of RNA into exosomes (Skog et al., 2008; Valadi et al., 2007). Previously, they demonstrated that exosomal RNAs share specific sequence motifs that may potentially duty as cis-acting elements for targeting to exosomes (Batagov, Kuznetsov & Kurochkin, 2011).

Given the fact that exosomes carry tangled biological information consisting of proteins, lipids and RNAs, it is not surprising to find that they gain been implicated in a variety of physiological and pathological conditions. Skokos et al. (2001) reported, for example, that mast cells communicate with other cells of the immune system via exosomes promoting mitogenic activity in B and T lymphocytes. A number of studies gain demonstrated the role of exosomes in the progress of the nervous system, synaptic activity, neuronal regeneration, neuron-glia communication and protection against injury (Lai & Breakefield, 2012). Furthermore, exosomes can breathe involved in the pathogenesis of cancer and degenerative diseases. The fact that tumor cells release a big amount of exosomes was initially demonstrated in ovarian cancer patients (Taylor, Homesley & Doellgast, 1983). Exosomes were shown to breathe secreted by various tumor cells including those derived from breast (King, Michael & Gleadle, 2012), colorectum (Silva et al., 2012), brain (Graner et al., 2009), ovarian (Escrevente et al., 2011), prostate (Mitchell et al., 2009; Nilsson et al., 2009), lung (Rabinowits et al., 2009), and bladder (Welton et al., 2010) cancer. A significantly higher amount of exosomes was create in plasma from lung cancer patients compared to that of control individuals (Rabinowits et al., 2009). In colorectal cancer patients, the amount of plasma circulating exosomes was constitutively higher than in routine healthy individuals showing the direct correlation between exosomes quantity and malignancy (Silva et al., 2012).

Manipulating tumor cells to abate the release of exosomes followed by their injection into immunocompetent mice led to a significantly slower tumor growth compared to that of unperturbed cells (Bobrie et al., 2012). It has been shown that exosomes which are released from tumor cells are able to transfer a variety of molecules, including cancer-specific, to other cells (Al-Nedawi, Meehan & Rak, 2009; Muralidharan-Chari et al., 2009) to maneuver their environment, making it more conducive for tumor growth and invasion. Glioblastoma-derived exosomes were create to breathe enriched in angiogenic proteins that allowed them to stimulate angiogenesis in endothelial cells (Skog et al., 2008). Melanoma exosomes were shown to breathe instrumental in melanoma cell dissemination via alterations in the angiogenic microenvironment. Hood et al. demonstrated that metastatic factors amenable for the recruitment of melanoma cells to sentinel lymph nodes are upregulated by melanoma exosomes themselves (Muralidharan-Chari et al., 2009). Exosomes shed by human MDA-MB-231 breast carcinoma cells and U87 glioma cells were capable of conferring the transformed characteristics of cancer cells onto routine fibroblasts and epithelial cells in share due to transferring tissue transglutaminase and fibronectin (Hood, San & Wickline, 2011).

Because of their small size exosomes gain an skill to penetrate intercellular contacts to attain far-off parts of the cadaver with the aid of the blood stream and other cadaver fluids. Exosomes gain been purified from human plasma, serum, bronchoalveolar fluid, urine, tumoral effusions, epididymal fluid, amniotic fluid and breast milk (Raposo & Stoorvogel, 2013). Since exosomes possess characteristic protein and RNA signatures of their host cells, analysis of exosomes in various cadaver fluids can breathe potentially utilized for non-invasive diagnostics of cancer and other disorders. For example, aggressive human gliomas often express a truncated and oncogenic shape of the epidermal growth factor receptor, known as EGFRvIII. The tumour-specific EGFRvIII was detected in serum microvesicles from glioblastoma patients (Skog et al., 2008). high stability of exosomal RNA (Skog et al., 2008; Valadi et al., 2007) and ease of RNA detection by highly sensitive PCR makes detection of exosomal RNA an attractive approach for the discovery of biomarkers. Indeed, mRNA variants and miRNAs characteristic of gliomas could breathe detected in serum microvesicles of glioblastoma patients (Skog et al., 2008). Expression profiles of serum microvesicle mRNA by microarrays correctly separated individuals with glioblastoma from routine controls (Noerholm et al., 2012). Of each and every RNA species, secreted miRNAs were most frequently utilized toward discovery of cadaver fluid-based biomarkers perhaps because miRNA expression profiles are more informative than mRNA expression profiles in a number of diseases (Grady & Tewari, 2010). In one study, analysis of plasma- and serum-derived microvesicles revealed 12 miRNAs differently expressed in prostate cancer patients compared to that of healthy controls and 11 miRNAs upregulated in patients with metastases compared to that of patients without metastases (Bryant et al., 2012). Interestingly, exosomes released by breast cancer cells can breathe separated into different classes depending on their miRNAs content (Palma et al., 2012). Cells undergoing malignant transformation produced exosomes containing selective miRNAs, whose release is increased by malignant transformation, in contrast to cells that are not affected by malignancy, whose exosomes are packed with neutral miRNAs (Palma et al., 2012). The changes in exosomal miRNA cargo could provide a signature of the presence of malignant cells in the body.

The majority of reported to date exosomal miRNA and mRNA profiles gain been generated using microarray approaches that suffer from several limitations. Microarrays are biased for investigation of already discovered transcripts. In addition, there is potential for cross-hybridization of RNAs that are highly related in sequence. Recently developed next generation RNA sequencing technology (RNA-Seq) allows detection of each and every RNA subtypes as well as of unannotated transcripts and has a high sensitivity toward identification of low-abundance RNAs. In case of exosomes, this approach was applied only for the analysis of small (20–70 nt) RNAs (Bellingham, Coleman & Hill, 2012; Nolte-’t Hoen et al., 2012).

In this study, they utilized the RNA-Seq approach to characterize the transcriptomes of exosomes secreted by two metastatic human breast cancer cell lines. They depict optimized computational workflow to analyze data generated by the Ion Torrent semiconductor chip-based technology. They gain identified and profiled RNA species present in exosomes and host cells and discuss the utility of exosomal RNA as potential breast cancer-specific biomarkers.

Materials and Methods Cell culture

Human breast cancer cell lines MDA-MB-436 (ATCC® HTB-130™) and MDA-MB-231 (ATCC® HTB-26™) were maintained at 37°C in 5% CO2 and cultured in DMEM/F12 supplemented with 10% FBS. 48 h prior to exosome collection, cells were washed 3 times with PBS and the medium was changed to serum-free CCM5 medium (Thermo Scientific).

Exosome extraction

Exosomes were isolated and purified from the media of MDA-MB-436 and MDA-MB-231 cell cultures using sequential centrifugation protocol. Briefly, media was collected and cellular debris was removed by centrifugation at 3,000×g for 10 min. The supernatant was centrifuged at 17,000×g for 30 min at 4°C. The supernatant was collected and centrifuged at 100,000×g for 2 h to pellet exosomes. Exosomes pellets were then washed in filtered PBS and re-centrifuged at 100,000×g, the supernatant was removed and the final exosomal pellet was re-suspended in 100 µl PBS.

Transmission electron microscopy

A 50 µl aliquot of exosomes was absorbed onto formvar carbon coated nickel grid for 1 h. The grid was positioned with the coating side facing the drop containing exosomes. Then the grids were washed by sequentially positioning them on top of the droplets of 0.1 M sodium cacodylate, pH 7.6 and then fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.6 for 10 min. Then grids were washed again with 0.1 M sodium cacodylate, pH 7.6 and contrasted with 2% uranyl acetate in 0.1 M sodium cacodylate, pH 7.6 for 15 min. After washing, the grids were incubated on top of the drop of 0.13% methyl cellulose and negatively stained with 0.4% uranyl acetate for 10 min, air dried for 5 min and examined with a JEM-2200FS transmission electron microscope operated at 100 kV.

Nanoparticle tracking analysis

Supernatants containing vesicles were analyzed using a Nano-Sight LM10 instrument equipped with a 405 nm laser (NanoSight, Amesbury, UK) at 25°C. Particle movement was tracked by NTA software (version 2.2, NanoSight) with low refractive index corresponding to cell-derived vesicles. Each track was then analyzed to rep the mean, mode, and median vesicle size together with the vesicle concentration (in millions) for each size.

RNA isolation and analysis

Total RNA from exosomes (MDA-MB-436 and 231) and cultured cells (MDA-MB-231) were isolated using the TRIzol reagent (Invitrogen). RNA character and concentration were assessed with the Agilent 2100 Bioanalyzer (Thermo Scientific). Cellular RNA was analyzed using RNA 6000 Nano Kit (Agilent) and exosomal RNA was analyzed using RNA 6000 Pico kit (Agilent).

RNA-seq with ion torrent personalized genome machine (PGM)

Two hundreds ng of exosomal RNA and 3 µg of total cell RNA was used as the starting input for RNA-Seq library preparation. Sequencing was performed by AITbiotech company (Singapore). Briefly, total cell RNA was treated with RiboMinus Eukaryote kit (Life Technologies) to remove rRNA. Then, exosomal and rRNA-depleted cellular RNAs were fragmented using RNaseIII. gross transcriptome library was constructed using the Ion Total-RNA Seq Kit v2 (Life Technologies). Bar-coded libraries were quantified with qRT–PCR. Each library template was clonally amplified on Ion Sphere Particles (Life Technologies) using Ion One touch 200 Template Kit v2 (Life Technologies). Preparations containing bar-coded libraries were loaded into 318 Chips and sequenced on the PGM (Life Technologies).

cDNA synthesis and quantitative real-time PCR (RT-PCR)

Two-step RT-PCR was performed using the QuantiTect reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) according to manufacturer’s protocol. RT-PCR was performed in a Rotor-Gene (Qiagen) using a SYBR Green PCR Master Mix. Primer sequences are provided in Table S3. each and every reactions with template and without template (negative controls) were race in duplicate and averaged. GAPDH was used as internal control for mRNA. Ct value was detected for each gene acceptation the cycle number at which the amount of amplified gene of interest reaches a fixed threshold. Relative quantification (fold change) was determined for the host cells and exosomal genes expression and normalized to an endogenous control GAPDH relative to a calibrator as 2−ΔΔCt (where ΔC = (Ct of gene of interest) – (Ct of endogenous control gene (GAPDH) and ΔΔCt = (ΔCt of samples for gene of interest) – (ΔCt of calibrator for the gene of interest). Melting curves of each amplified products were analyzed to ensure uniform amplification of the PCR products.

Bioinformatics analysis Raw reads filtering

Raw reads generated by sequencing were subjected to several character checks. The low character reads were removed by read trimming and read filtering. Read trimming included removal of adapter sequences, removal of the 3′ ends with low character scores and trimming based on High-Residual Ionogram Values. Filtering of entire reads included removal of short reads, adapter dimers, reads lacking sequencing key, reads with off-scale signal and polyclonal reads. Subsequent analysis was performed with high character reads which passed through the described above filtering steps.

Reads mapping

Bowtie 2 version 2.1.0 was used to align each and every high-quality reads to rRNA sequences including 28S (NR_003287.2), 18S (NR_003286.2), 5S (NR_023379.1), and 5.8S (NR_003285.2) rRNA. Reads mapped to rRNA sequences were filtered out while the relaxation of the reads were mapped to the human genome. The high-quality reads were mapped to hg19 build of the human genome from University of California Santa Cruz (UCSC) genome browser database (Meyer et al., 2013) using TopHat version 2.0.6 with the aligner Bowtie 2.0.5 (Kim et al., 2013; Langmead & Salzberg, 2012) with their default parameters in end-to-end mode (-b2-sensitive) and defining splice-junctions based on known splice-junctions (-G). To classify the reads into known and unknown genes, the BAM file generated by Tophat2 was intersected to known gene (RefGene and GENCODE built V14 from UCSC database) using BEDtools (Quinlan & Hall, 2010) and was used to signify the number of reads by SAMtools (Li et al., 2009).

Post-processing of the aligned reads

The mapped reads were further manipulated by removing the reads that mapped to multiple locations. In particular, the short aligned reads with the length of <20 nucleotides were eliminated to avoid the alignment errors such as mapping to multiple genomic locations. Further filtering included the removal of the low character reads which tumble below the mapping character score of 10 (-q 10) using SAMtools. For the coverage search, the BAM file generated by Tophat2 was converted to BED format with option (-split) using BEDtools. The BED file was converted again to BAM format using BEDtools. They then developed python script (using pysam as share of the scripts) to cipher the number of reads and read coverage in exons and protein-coding sequence (CDS) regions consecutively.

RNA abundance calculation

RNA abundance was estimated with the aid of Partek Genomics Suite software (Partek Inc., St. Lous, MO) using Reference Sequence Gene (RefSeq Gene) and GENCODE annotation built version 14 of those not overlapped with RefSeq Gene from UCSC genome browser. The Expectation-Maximization (E/M) Algorithm (Xing et al., 2006) was used to estimate the most likely relative expression levels of each gene isoform. Partek’s algorithm was used to quantify the gene isoform expression level as reads per kilo groundwork per million mapped reads (RPKM).

Functional analysis of genes

Database for Annotation, Visualization, and Integrated Discovery (DAVID) (version 6.7) was used to identify gene functional annotation terms that are significantly enriched in particular gene lists with the gross Human genes as the background (Huang, Sherman & Lempicki, 2009). A list of gene symbols was generated for each dataset and was used as input into DAVID. DAVID calculates a modified Fishers Exact p-value to demonstrate Gene ontology (GO) and KEGG molecular pathway enrichment, where p-values less than 0.05 after Benjamini multiple test correction are considered to breathe strongly enriched in the annotation category. They besides used a signify threshold of 5 and the default value of 0.1 for the EASE (enrichment) score settings. They used more specific proceed term categories provided by DAVID, called proceed FAT, to minimize the redundancy of universal proceed terms in the analysis to increase the specificity of the terms.

Results Characterization of exosomes released by breast cancer cells

Exosomes were isolated from two breast cancer cell lines, MDA-MB-436 and MDA-MB-231 using classical ultracentrifugation protocol. The size distribution and amount of exosomes were analyzed using NanoSight LM10 nanoparticle tracking analysis (NTA). NTA showed that MDA-MB-231 cells released 4 × 106 vesicles per cm2 of growth locality per 48 h that were predominantly 115 nm in size. MDA-MB-436 cells released 1.04 × 107 vesicles per cm2 of growth locality per 48 h that were predominantly 91 nm in size (Fig. 1). The size of exosomes released from both cell lines ranged from ∼70 nm to ∼300 nm. An examination of the purified vesicles using transmission electron microscopy revealed that they had the size (∼50–100 nm) and morphology (Fig. 2) typical of that of exosomes.

Figure 1: Analysis of exosomes produced by breast cancer cell lines, MDA-MB-436 and MDA-MB-231, with Nanosight LM10-HS instrument. Characterization of exosomal RNA

RNA was isolated from exosomes released by both breast cancer cell lines. Total RNA was besides extracted from MDA-MB-231 cell line as a control host cell line that produced exosomes. Bioanalyzer data revealed that exosomes accommodate a broad range of RNA sizes (30–500 nt) and gain very small amount of intact rRNA (5.2% in MDA-MB-231 exosomes and 5.6% in MDA-MB-436 exosomes) (Fig. 3) consistent with previous reports on exosomal RNA (Rabinowits et al., 2009; Valadi et al., 2007).

Figure 2: TEM image of the exosomes produces by MDA-MB-436 cell line. Electron microscopy allowed visualizing membrane-bound nanovesicles sized ∼100 nm. White arrowheads pointing to the exosomes. Scale bar = 100 nm. Figure 3: Analysis of RNA from cells and exosomes by Bioanalyzer. Exosomal and total cell RNA was analyzed with PicoChip and NanoChip, respectively. Exosomal RNA profound sequencing using Ion Torrent technology

We utilized the Ion Torrent sequencing technology to profile exosomal RNA produced by MDA-MB-436 and MDA-MB-231 cell lines, as well as RNA obtained from host cell line MDA-MB-231. Based on RNA character assessment using Bioanalyzer profiling (Fig. 3) they performed rRNA depletion for cellular RNA but not for exosomal RNA.

Identification of appropriate alignment tool for efficient capture of splice-junction reads produced by the Ion Torrent technology

RNA-Seq computational analysis workflow is presented in Fig. 4. The single-end RNA reads generated from sequencing of the exosomal and host cell libraries were trimmed to remove adapter sequences and then filtered. After filtering out low-quality reads, the high-quality reads had a varying length ranging from 6 bp to >300 bp. These preceded high-quality reads were considered for further analysis. The total amount of reads for RNA from MDA-MB-231 cells was about 4.8 M. RNA-Seq resulted in ∼3.5 M and ∼3.2 M reads for RNA from MDA-MB-436 and MDA-MB-231 cells derived exosomes, respectively. At first, the reads were aligned to rRNA sequences including 28S, 18S, 5S, and 5.8S rRNA (see Methods). 24% of the reads in cellular and more than 80% of the reads of both exosome samples were mapped to rRNA sequences (Fig. S2). The relaxation of the reads were mapped to the human genome (UCSC; hg19 built) with the aid of TMAP aligner program in accordance with the Ion Torrent recommended pipeline (Technologies) (data not shown). However, the use of TMAP resulted in misalignment of the splice-junction reads or reads containing multiple exons to the genome (Fig. S1). Therefore, they used alternative aligner Tophat 2.0.6 along with the Bowtie 2.0.5 for the read mapping.The mapped results are shown in Fig. S2. In total, this alignment produced more than 4.3 M (∼90%) reads of MDA-MB-231 cellular RNA that could breathe mapped to rRNA sequences and the human genome. For the MDA-MB-231 and MDA-MB-436 cell-derived exosomes, these alignments resulted in ∼2.8 M (∼90%) and ∼3.2 M (∼91%) of mapped reads, respectively (Fig. S2). More than 80% of mapped reads from exosomal samples were create to breathe mapped to rRNA sequences. They further investigated the reads of both exosomal RNA samples, which mapped to rRNA sequences. These reads were counted and plotted as read density over each rRNA sequence (shown in Fig. S3A). The mapped reads covered entire length of each and every rRNA sequences. The major fractions of mapped reads were 28S and 18S rRNA (Fig. S3B).

Figure 4: Flowchart of RNA-seq data analysis. The raw reads are exposed to pre-alignment character checks including the removal of adaptor sequences and low character reads. The high character reads are then mapped to rRNA sequences using Bowtie2 version 2.1.0. The non-mapped rRNA reads were mapped to human genome hg19 build of the human genome using TopHat version 2.0.6 with the aligner Bowtie 2.0.5. After mapping, low mapping character reads less than 10; short reads with length less than 20 groundwork pairs and multi- reads were removed. Estimation of read counts and read coverage on mapped reads where over 90% of exon (non-coding) and CDs (for coding) in transcript isoforms of RefGene and/ or GENCODE v14 gene models. The EM algorithm along with GENCODE v14 annotations was used to estimate the read signify and reads per kilo groundwork per million mapped reads (RPKM) on mapped transcripts.

Reads mapped to multiple locations in the genome gain been eliminated to obtain uniquely mapped reads. Subsequent downstream analysis was performed with the high character reads which had the read length greater than 20 bps and mapping character score of above 10 (see Methods). As a result, about 81, 76, and 70% out of each and every mapped reads were considered as high character mapped reads for MDA-MB-231 and MDA-MB-436 exosomal RNA, and MDA-MB- 231 cellular RNA, respectively.

Approximately 97% (∼2 M reads) of reads from both exosomal RNA samples were mapped to rRNA sequences (Fig. 5) and ∼2% of the reads were mapped to known genes (RefSeq and/or GENCODE gene models). At the same time, for the MDA-MB-231 cellular RNA, for which rRNA depletion step was performed, the majority of the reads (∼58%) was mapped to known genes (Fig. 5). To increase the number of reads for other RNAs they attempted to impoverish rRNA with the RiboMinus™Eukaryote Kit recommended by Ion Total RNA-Seq Kit protocol. As expected, this protocol efficiently pulled down rRNA from cellular RNA (>60%) but had itsy-bitsy upshot on removal of rRNA from exosomal RNA (Fig. S4). The failure to impoverish exosomal fragmented rRNA can breathe explained by the design of the RiboMinus™Probe. It consists of 2 probes each for 5S, 5.8S, 18S and 28S RNA. Because the probe size is 22–25 nucleotides, many fragments of rRNA are not targeted.

Figure 5: Distribution of uniquely mapped RNA-seq reads among transcriptome. Reads which overlapped with annotated gene models (RefSeq and/or GENCODE) are termed as “known genes”. Reads that placed outside of annotated gene models are termed as “unknown”. Reads which are mapped to rRNA sequences including 5S, 5.8S, 18S, and 28S rRNA are named as “rRNA”. Content of cellular and exosomal transcriptomes

In total, 16,086 transcripts (11,657 genes) were detected with a normalized RPKM value of greater than 1.0 at least in one sample. They used Integrative Genomics Viewer (IGV) version 2.1.21 (Thorvaldsdottir, Robinson & Mesirov, 2013) to visualize mapped reads and to check the coverage across human transcriptome. They observed frequently misannotated transcripts in exosome samples with high RPKM value in those cases when only small parts of the transcripts were covered by the reads (Fig. 6). This was the upshot of low depth sequencing caused by the presence of a big amount of reads representing rRNA. Therefore, they implemented more stringent criteria to obtain full-length expressed genes by filtering genes based on the RNA reads coverage. Reads which cover over 90% of protein-coding sequence (CDS) of protein-coding genes and over 90% of exons in non-coding sequence of non-coding genes were considered for further analysis. To identify protein-coding genes in exosomal samples, the mapped reads of exosomal RNA were pooled with estimated CDS coverage of >90% for both exosomal reads. The pooled mapped reads were used to cipher CDS in each exosomal sample as >50% of coverage.

Figure 6: specimen of low coverage transcript but very high RPKM in AURKAIP1 and ATPIF1 genes. (A) AURKAIP1 gene from chromosome position chr1:1,309,009–1,310,847 is shown using Integrative Genomic Viewer. Among the three variants, the maximum value of protein-coding sequence (CDS) coverage, read signify and RPKM is shown in the right panel of read mapping. Both the exosomes shows very low coverage (7–22%) with read counts of 4, whereas the RPKM value is 65.44 and 80.78 RPKM for exosomes of MDA-MB-231 and MDA-MB-436, respectively. (B) ATPIF1 gene from chromosome position chr1:28,562,494–28,564,655 is visualized. The MDA-MB-231 exosomes exhibit high CDS coverage (91%) with an exon signify of 4.

Using these criteria, they obtained lower number of annotated transcripts (6,187 transcripts or 3,437 genes) compared to that when RPKM values were considered (Fig. S5 and Table S1). As a result, some transcripts showed high coverage with CDSs criteria (for example, ATPIF1) in exosomal RNA from MDA-MB-231 cells, even when the number of reads was small (less than 5) (Fig. 6B). Such genes were besides taken into consideration in their analysis. In total, 5821 (3115 genes) and 187 (115 genes) protein-coding transcripts were detected based on the RNA reads coverage in cellular and exosomal samples, respectively. For non-coding genes, 360 (317 genes) and 131 (131 genes) transcripts were detected based on the RNA reads coverage for cellular and exosomal samples, respectively. Analysis of these transcripts revealed that they represented 90.8% of protein-coding genes and 9.2% of non-coding genes for host cell sample; while exosomal RNA samples represented 50.4% and 49.6% of protein-coding and 47.6% and 52.4% of non-coding genes in MDA-MB-231 and MDA-MB-436 cells derived exosomes, respectively (Table 1). They create that 98.3% of protein-coding and 97.7% of non-coding exosomal transcripts were present in host cells (Fig. 7).

Table 1:

Amount of genes in cellular and exosomal RNA based on 90% coverage over protein-coding sequence of genes and exons of non-coding genes.

Note the big balance of non-coding transcripts in exosomal RNA. Transcript type MDA-MB-231cellulargenes (%) MDA-MB-231exosomalgenes (%) MDA-MB-436exosomalgenes (%) Protein-coding 90.8 50.4 47.6 Non-coding 9.2 49.6 52.4 Figure 7: Venn diagram presents overlap among protein-coding and non-coding gene symbols in exosomes and cells. Almost each and every the genes in both exosomal RNA are the subset of cellular genes. Exosomes are enriched in mRNAs functioning in protein translation and rRNA processing

We performed Gene Ontology (GO) enrichment analysis using the DAVID bioinformatics resource, which employs a Fisher’s Exact Test with Benjamini–Hochberg correction. A total of 377 enriched proceed categories were derived using a P-value cut-off of p < 0.05 for 3115 host MDA-MB-231 cellular genes: 286 Biological Process (BP) categories and 91 Molecular duty (MF) categories (Table S2). In total, 18 proceed categories including 11 BP and 7 MF were derived from 115 exosomal genes from both cell lines. figure 8 shows top 20 BP categories of the host cellular genes which comprehend translation process, cell cycle, RNA processing, etc. (Fig. 8A and Table S2B). At the same time exosomal genes revealed biological processes in translation, ribosome biogenesis, rRNA and ncRNA processing proceed categories (Fig. 8B and Table S2C). Since the major fraction of exosomal samples were rRNA species, significantly lower number of mRNA could breathe detected in exosomal samples. They hypothesized that the genes detected from exosomal samples should breathe highly expressed in the cells. To test the hypothesis, they performed proceed enrichment analysis for 115 top expressed genes from MDA-MB-231 cellular sample. The top 10 proceed terms (Fig. 8C and Table S2D) of these top expressed genes are the same as in exosomal fraction (Fig. 8B and Table S2C). They further created box plot of 115 exosomal genes in MDA-MB-231 cellular sample using expression values (RPKM) (Fig. 9). These data clearly showed that exosomes are enriched in genes which are highly expressed in the host cells.

Figure 8: Gene Ontology (GO) enrichment analysis of genes detected in cellular and exosomal RNA from breast cancer cell lines. The significant proceed terms was defined as described in Materials and Methods. (A) Top 20 significant proceed terms create in MDA-MB-231 cellular genes (3115 genes). (B) Significant proceed terms create in exosomal genes from both cell-lines (MDA-MB-231 and MDA-MB-436). (C) Top 20 significant proceed terms create in the most expressed 115 genes from MDA-MB-231 cellular genes. The asterisks (*) argue proceed terms that present in exosomal genes. Figure 9: Expressed genes in exosomes create to breathe highly expressed in the host cells. The box plot indicates expression level of each and every genes in cellular samples as compared to that of genes which were create to breathe express in exosomes. Wilcoxon rank sum test represents significant incompatibility in expression level of the two sets.

Non-coding transcripts could breathe classified into 13 categories (see Table 2). Both exosomal and cellular samples contained small nucleolar RNA (snoRNA) as major species. The second most abundant class of non-coding transcripts according to GENCODE annotation was “non-coding RNA” in cellular sample and small nuclear RNA (snRNA) in exosomal samples. Overall, the top five RNA categories represented about 90% of each and every noncoding genes in both exosomal and cellular RNA.

Table 2:

Amount of non-coding gene attribute in cellular and exosomal RNA based on 90% coverage over exons of non-coding transcripts.

In exosomes, the top 5 non-coding gene types including small nucleolar RNA, small nuclear RNA, Mt_tRNA, microRNA, and non-coding RNA represents about 90% of non-coding genes in both exosome samples. Gene type MDA-MB-231cellular(gene symbols) MDA-MB-231exosomal(gene symbols) MDA-MB-436 exosomal(gene symbols) small nucleolar RNA 214 83 51 small nuclear RNA 23 11 10 Mt_tRNA 13 7 4 microRNA 34 6 2 non-coding RNA 42 1 0 guide RNA 20 0 1 vault RNA 3 0 3 rRNA 1 1 2 RNase MRP RNA 1 1 1 RNase P RNA 1 1 1 Mt_rRNA 1 1 0 lincRNA 1 0 0 telomerase RNA 1 0 0 Validation analysis of RNA-seq data by qRT-PCR

Based on RNA-Seq data they evaluated presence and enrichment of several mRNA transcripts in exosomal RNA - RAB13, RPPH1, EEF1A1, FTH1, FTL and RPL28. qRT-PCR analysis showed presence of each and every selected transcripts in exosomal samples (Fig. 10A). figure 10B demonstrates that the fold-change of qRT-PCR results are consistent with the fold-change of RNA-seq data.

Figure 10: Validation of RNA-seq data by qRT-PCR. (A) Ct values for six mRNA transcripts which were detected in exosomal samples by RNA-seq are shown. (B) Comparison of different expression values (RPKM; MDA-MB-436/RPKM; MDA-MB-231) detected by RNA-Seq (dark-grey columns) and qRT-PCR (light-grey columns) for six differently expressed genes. Discussion

Until recently, the changes in gene expression during various biological processes gain been analyzed using microarray approaches that focus largely on the conduct of protein-coding transcripts. Because microarrays are based on hybridization, they gain high background owing to cross-hybridization, they gain a limited dynamic range of detection and they reckon upon known structures of genes. progress of RNA-Seq technology permitted comprehensive analysis of gross transcriptomes with the sole nucleotide resolution allowing quantification of most RNA molecules expressed in the cell or tissue (Mortazavi et al., 2008). In this study, they used the Ion Torrent platform to interrogate transcriptomes of exosomes released from two metastatic breast cancer cell lines. At the time of conducting their analysis this technology produced relatively low number of reads, yet they selected it as it provided the longest reads than any other sequencing platform. This feature of the Ion Torrent technology was essential as they dealt with RNA isolated from exosomes whose nature and composition are quiet not well established. RNA-seq data analysis is complicated by the intricacy of dealing with big datasets, reads character control, alignment procedure etc. Different workflows and several algorithms gain been proposed to map reads to the reference genome and to effect data analysis (Chen, Wang & Shi, 2011; Mortazavi et al., 2008). Comparison of expression levels across different samples and experiments is often difficult and requires complicated normalization methods and these are quiet under vigorous development. The situation is even more tangled in case of exosomal transcriptomes that vary significantly from cellular transcriptomes.To address this issue, they developed in this study customized bioinformatics workflow and demonstrated its utility for analysis of exosomal RNA. Because the Ion Torrent platform produces reads with different length the dedicated algorithm for their alignment to the genome called TMAP was recommended. They create out, however, that this tool does not allow satisfactory mapping of reads that accommodate splice-junctions or span introns. Therefore, they elect alternative aligning tool TopHat2 (with Bowtie2) which could ply reads of varying length and identify splice-junctions based on known splice-junctions as well as allowed the discovery of current splice-junctions (Kim et al., 2013; Langmead & Salzberg, 2012).

We observed a big balance of reads mapped to rRNA regions in exosomal samples. This was surprising given the fact that intact 18S and 28S rRNA peaks were almost undetectable in exosomal RNA (Fig. 3). This observation suggested that the majority of exosomal rRNA is fragmented. Exosomal rRNA fragments could breathe mapped over entire length of rRNA (Fig. S3). Fragmented 28S and 18S rRNA were major rRNA species present in exosomes. The reads mapped to 28S and 18S rRNA were distributed almost equally in exosomal and cellular RNA samples. What is the feasible understanding for generation of exosomal rRNA fragments? RNases present in cell culture conditioned medium are unlikely to contribute to rRNA fragmentation since exosomal membranes provide protection against RNase attack. Indeed, treatment of the exosomal preparations with RNase A did not lead to significant incompatibility between treated and control samples in RNA size distribution (data not shown). In the study of Skog et al. (2008) RNase treatment of the glioblastoma exosomes led to a very insignificant (less than 7%) abate in RNA suggesting that exosomal RNA is inaccessible for RNase from outside the vesicles. A possibility exists that the rRNA fragments are generated after secretion by RNases originated from the host cells and incorporated into exosome vesicles. Alternatively, rRNA fragments could breathe generated inside cells prior to their release to exosomes. Another class of RNA, tRNA is represented in exosomes mainly by its fragments (Nolte-’t Hoen et al., 2012). The most abundant tRNA hits in exosomal RNA are each and every located at the 5′ quit of ripen tRNAs (Nolte-’t Hoen et al., 2012).

Regardless the biogenesis of rRNA fragments, it is advisable to effect rRNA depletion step even in the absence of visible rRNA peaks on RNA profiles. This procedure would allow obtaining much higher sequencing depth for other RNA species. Their attempt to impoverish fragmented rRNA with the favorite RiboMinus™Eukaryote Kit failed because of the design of the probes. Because the probes size is short, many fragments of rRNA are not targeted. The use of larger number of longer probes is expected to submit a more efficient way of pulling-down fragmented rRNA. This technical aspect of working with exosomal RNA samples should breathe certainly considered in the future studies.

As a result of big rRNA presence in exosomal samples they observed only 2% of mapped reads to known transcripts using RefSeq and GENECODE gene models. Moreover, with RPKM value >1 they observed a big amount of misannotation due to poor coverage of the reads over transcripts. Therefore, they suggested another approach, namely filtering genes based on reads coverage over protein coding sequence for mRNA or exons for non-coding RNA. This procedure allowed us to achieve more than 90% coverage for protein-coding and non-coding regions which they considered as highly amenable for functional classification. This approach was helpful to expose highly expressed genes in exosomes which could breathe potentially used as noninvasive breast cancer markers.

We report that exosomes are carrying mRNAs that are highly expressed in the host breast cancer cells (Fig. 9). Thus exosomal transcriptomes are representative of their cells of inception and should provide a platform for detection of tumor specific markers. proceed analysis revealed that main biological and molecular functions of both cellular and exosomal transcripts are enriched in proteins involved in ribosome biogenesis, translational elongation, ribosomal subunit assembly and rRNA processing. What could breathe the significance of these functions in exosomal transcriptome? Exosome-associated mRNAs were shown to breathe translated into proteins by recipient cells (Ratajczak et al., 2006; Valadi et al., 2007). They hypothesize that upon arrival to the recipient cells exosomal mRNAs are translated into proteins supporting ribosomal functions to ensure efficient translation of other exosomal mRNAs within a cellular compartment where exosome content is released. Valadi et al. (2007) besides described presence of mRNAs for many ribosomal proteins in exosomes secreted by mouse mast cell line. Interestingly, Graner et al. (2009) demonstrated the presence of elongation and translation factors in exosomes derived from brain tumor.

In conclusion, here they demonstrated for the first time that fragmented rRNA is a major species of exosomal RNA. Proposed here custom bioinformatics workflow allowed us to reliably detect other, non-ribosomal RNAs under conditions of limited read numbers. Classification and quantification of the RNA-Seq data revealed that exosomal transcripts are representative of their cells of inception and thus could shape basis for detection of tumor specific markers. This information can besides breathe used for getting insights in the molecular underpinnings of biological effects produced by these microvesicles. Finding that exosomes tolerate mRNAs encoding the necessary components to build-on-site ribosomes provides a valuable insight into biological duty of these vesicles.

Supplemental Information Comparison of mapping character between the alignment tools TopHat2.0.6 and TMAP 0.3.7

FTL gene (chromosome position chr19:49,468,467-49,470,296) is selected as an specimen of alignment comparison. TMAP alignment resulted in poor reads mapping and absence of junctions over exon-exon region. As the same time, TopHat identifies the exon-exon splice junctions and connects the exons through a linker.

Distribution of each and every mapped and unmapped RNA-seq reads among genomic compartments

rRNA defined as 5S, 5.8S, 18S, and 28S rRNA sequences. Reads which overlapped with annotated gene models (RefSeq and/or GENCODE) are termed as “known genes”. Reads that placed outside of annotated gene models are termed as “unkown”.

Fragments of rRNA in exosomes depict full-length of rRNA sequence

(A) RNA read density plot represents RNA fragments which fully covers of 5S, 5.8S, 18S, and 28S rRNA sequences from exosomal RNA. (B) 18S and 28S rRNA were major fractions of rRNA species.

Analysis of rRNA depletion from MDA-MB-231 cellular and exosomal RNA using RiboMinus™ Eukaryote Kit for RNASeq

RNA was detected with PicoChip using Bioanalyzer. The depletion procedure has been performed according to the manufacturer’s protocol. Control samples (red) were treated exactly as experimental samples (blue) except they did not accommodate RiboMinus™ Probe.

Venn diagram of genes generated by RPKM and read coverage approaches

The detection criteria is that gene has more than 1 RPKM in at least one sample, while another approach is that gene has more than 90% coverage over protein-coding or non-coding sequence.

Two approaches of gene transcripts selection using RPKM or read coverage

(A) The Partek genomic suite output showing transcripts with RPKM >1 in at least one sample. The read counts from the transcript isoforms were estimated using EM algorithm from Partek genomic suite on RefGene and/or GENCODE v14 gene models. (B) Estimation of read coverage (in percentage) and read signify of transcript. The transcripts with >90% coverage for protein-coding sequence and exonic sequence (in case of non-coding transcript) of transcript isoforms on RefGene and/or GENCODE v14 gene models are shown.

Gene Ontology (GO) enrichment analysis using the DAVID bioinformatics resource of Genes create in cellular and exosomal samples

(A) Gene lists for proceed enrichment analysis. (B) proceed enrichment of cellular genes. (C) proceed enrichment of exosomal genes. (D) proceed enrichment of top 115 expressed cellular genes.

List of primers used for qRT-PCR

How efficient is Your Test Automation? | killexams.com existent questions and Pass4sure dumps

Do you know, for sure, absolutely, that your automated test is testing what you assume it's testing? Probably not, now that we've introduced some doubt. Mike Kelly explains how to rep from "I assume it's working" to "Yes, it's working correctly."

Like this article? They recommend 

How many times gain you been asked, "How attain you know that automated test is actually testing what you assume it is?" I've been asked that question a lot, and I gain even asked it myself on occasion. It's one of the classic issues with test automation: If no human is involved, how attain you really know that your tests are testing what you told them to test? What if there's a bug in your test code or in the test tool? What if the test wasn't implemented properly? What if a change in the test code in one Place causes code to fail in another? Maybe the test code is outdated due to changes in the application and is no longer testing anything.

You rep the point.

This article takes two approaches to these questions. First, we'll eye at some methods of validating that a test is in fact testing what it's putative to breathe testing. Then we'll consider what you can attain if you eye under the hood and find problems, and how some of these issues might breathe avoided. We'll focus on traditional scripted regression tests and basic performance tests.

I know of a brace of ways to validate automated tests. This is by no means an exhaustive list; these are just methods I've used in the past that gain worked (or failed) for me. They may not breathe practical in your context, or they may not drudgery for your software, but it's at least a Place to start.

Manual Reviews

Option 1, the agreeable old-fashioned code review. In a review, an automated test (functional or performance) is examined for accuracy by walking through the code and the log files, and by actually watching the test execute. You might even race the test in debug mode (if that's an option with your tool) and step through the code as it executes. This technique gives you the most visibility into what the script is actually doing, and it besides might breathe easier then trying to read the code without really knowing what it's putative to breathe doing.

Manual reviews require a inescapable level of familiarity with the automation tools, languages, any frameworks developed, and the expostulate of the test script being reviewed. This may not breathe an light set of knowledge to Come across. And if you attain gain staff who can effect the review, most likely they won't gain time to attain it because they're amenable for implementing current tests for the project team. In addition, this is a late (and sometimes painful) process, which requires a significant investment of person-hours in order to gain any existent assurance in the suite of tests.

Introducing Changes to the System Under Test

Another approach you can try is to insert specific changes into the system under test (sometimes called oversight seeding). For example, if I know I gain a sequence of automated tests that test the search feature on my web site, I might change the order in which results are returned and then race the scripts to ensure that they fail when they test that feature. With this approach, if the scripts pass, they're not testing what they should breathe testing. For performance tests, I might reconfigure my web server and execute the tests again, looking for change in the performance results.

While giving you better coverage than manual reviews, this system is not as reliable. In the examples I just described, some tests might fail with a different order on search results, but some tests might quiet pass because they don't check order but rather circumscribe conditions on the search field, or some other aspect that's not related to results. For the performance-testing example, you may change a server setting that has no noticeable impact to system performance; even though you assume it might (I've witnessed this). This means you quiet exigency to switch to manual reviews for those tests that passed when you thought they should gain failed.

In addition, now you're not taking just one person off routine project tasks for review, but several. Someone needs to add bugs to the code. Someone needs to build and deploy it (let's not talk about configuration-management issues). Someone needs to adjust the web server. Then you quiet gain the tester, who needs to execute and validate the tests. While this is probably faster than manual reviews, it's most likely much more costly and a lot less reliable.

Spinning Off a Short Side Project To Verify Something

In this approach, you simply validate your automated tests by duplicating them using some other tool or implementation model. This is more of a macro approach to the problem, while the other two gain been more micro in nature. I've only seen this done with performance tests, but I can imagine a brace of scenarios where it might shape sense to try it for functional automation. This system is probably the most costly, as it's a pellucid duplication of drudgery that has already been done, but sometimes this is the fastest way to gain assurance in something.

In his two-part sequence on performance tool comparisons (Part 1, share 2), Suresh Nageswaran shows an specimen of what this might eye like. In one of my current projects, we're trying to attain a similar exercise by verifying page-load times using both the Mercury and Rational performance test tools. Often, you only exigency to duplicate some small portion of the overall set of tests and then drudgery under the assumption that your implementation model is correct. The drawbacks to this approach are the cost of ownership for multiple tools and developing equivalent skills related to the tools and/or implementation models.

Overlapping Your Testing

This approach is probably my favorite, but it's difficult to find a context in which it can breathe implemented. In this approach, you simply attempt to audit one nature of testing (in this case, functional or performance test automation) by using another nature of testing. For example, I fondness to audit my performance tests whenever I race my functional tests. You can read about a specific specimen of this in my article "Gathering Performance Information While Executing Everyday Automated Tests." Similarly, you can sometimes attain the same thing by having your performance tests validate some sort of functional requirement (but this can breathe a bit more difficult and may breathe less practical). More traditionally, for projects in which I've planned their testing, I've tried to build in a small overlap on the manual test exertion and the automated test effort.

The costs for this approach are typically the lowest, as you're just tacking an extra job or two onto an existing effort. This means that no one stops what they're doing. It's your classic case of scope creep, only now you're attempting to use it to your advantage in testing. The disadvantage to this approach is that you now gain each and every the problems related to scope creep. The original job now takes a itsy-bitsy longer to finish, and someone has to use and maintain the overlapped functionality you developed. Don't rep too carried away with overlapping, as each time you attain it you're introducing inefficiency into the test project.



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