Psychology undefined, Darvin Essay Example | Topics and Well Written Essays - 500 words

Brain research indistinct, Darvin - Essay Example His Theory varies from Lamarckism in that Lamarck maintained a strategic distance from advancement, while Darwin's hypothesis remains legitimate whether gained attributes are transmitted or not, Lamarck's hypothesis becomes out of commission whenever procured characteristics can't be transmitted. Darwin's hypothesis is fundamentally censured for nonappearance of any solid observational logical proof on the side of the hypothesis, with respect to the unconstrained age of life or legacy starting with one then onto the next . Q2. Portray Galton's use of the hypothesis of advancement by normal determination to singular contrasts in human mental qualities. For example, what were Galton's suppositions about the idea of human mental qualities What sorts of proof did Galton acquire To what degree did the proof gathered by Galton offer solid logical help for his perspectives on singular contrasts and advancement of brain Answer: Galtons accepts that no creatures have ever been reproduced for general insight, as people we are having the high broad acumen that other living things haven't. Since no investigations had been made for this, yet we can completely perceive how discerning creatures are people. We are the most elevated type of living things. Galton made an examination having a true to life work that was an assortment of insightful people.

Filial Piety in China essays

Obedient Piety in China expositions Xiao, the Chinese word for obedient devotion is the characterizing highlight in Chinese culture as dutiful devotion was lauded as the most noteworthy goodness in China for quite a long time. I buy in to the way of thinking that obedient devotion is the foundation of Chinese morals and with everything edifying examinations appear. Chinese society was based upon the tenet of dutiful devotion and that it is the subject in Chinese culture, impacting all parts of Chinese lives. In this paper, I will look at the hugeness of dutiful devotion in Chinese culture. Yet I have no desire of committing the error of speculation, what I try to characterize is the inclination subject in every single Chinese family which I accept is obedient devotion. As I accept that obedient devotion is the consistent idea that runs in each Chinese family. I am completely mindful that my investigation of dutiful devotion doesn't have any significant bearing to each Chinese family, so I can just say that I am certain obedient devotion suffers in various structures, having been invaded through numerous ages and dissolved by various occasions. So as to make my stand, I will investigate the conventional importance of dutiful devotion in China, if and how different episodes may have affected obedient devotion in China. I will likewise be breaking down the ramifications of the disintegration of dutiful devotion and the significance of obedient devotion in present day China. To characterize conventional obedient devotion in Chinese culture, it is basic that I draw on crafted by Confucius as Confucianism is the framework that has commanded Chinese idea all through a large portion of history, controlling Chinese training, society and government for around 2,000 years. It is important to consider Mencius' way of thinking as he was likewise a solid effect on Chinese culture. Xiao in conventional Chinese social orders in a general sense implies appreciation to one's folks for giving one life and the obligation and commitment to compensate one's folks for having brought one up. It implies love and solid regard for one'... <!

To the Class of 2016

To the Class of 2016 After the chaos and confusion that was associated with MIT FPOPs (Freshman Preorientation Programs), orientation, and sorority recruitment, Im finally free to come back to the blogs (: Its not that I havent been thinking of them. In fact, I even wrote a little outline of this blog down on a napkin I just havent had a good chunk of time to sit down and actually type everything out. So here we go: Dear Class of 2016, Welcome to MIT! In the upcoming weeks, youll soon find yourself in the true swing of things at MIT. Classes. Psets. Exams. Eventually, youll learn the true meaning of IHTFP. MIT can be a wonderful or awful place depending on how you decide itll be. Heres just some things to keep in mind as you brave through your first semester here: Keep an open mind. There are some things that you hear before you come to MIT, and even while youre here, that might influence how you perceive things at the Tute. I remember as a freshmen hearing the stereotypes about dorms and greek life, and letting them initially prevent me from making my own decisions about different things on campus. But having been here for a year, you realize that theres always exceptions to the stereotype, and that things you never thought youd like are actually quite likeable. For that reason, keep an open mind. Youll never know what new opportunities might come out of the new things you try and the people you meet. Take care of yourself Sleep. Eat. Exercise. Its easy to let your health slip, especially when its snowing outside and youre stressed about all the work you have due the next week. Dont fall into the pithole though! Your health should be your first priority. If youre not feeling well, youre probably not going to be able to work your best anyways. So if youre tired, dont be afraid to take a nap (theyll become your new favorite pastime). If youre sick, dont be afraid to head over to MIT Medical or your floors MedLink. If youre feeling stressed, take a walk along the Charles or run at the Z center. Youll find youll be much happier if you take care of yourself during your time here.Dont forget to mentally take care of yourself either! By this I mean, make time for yourself. Whether its an hour bridge loop run, painting a picture, or playing your favorite instrument, make sure you have time to decompress. You time is never a waste of time. Youre not alone! During the thick of things, its easy to fall behind. But the important thing to remember is dont be afraid to ask for help. Often times, youll find that youre not the only one confused about specific topics.I remember my Spring semester, I struggled quite a bit. I took on a little too much, and because of all my commitments, I actually failed my first 5.111 test. But after I made it past all my emotions, I realized I needed to actually take action to fix my fifth week flag. I ended up scheduling an appointment with my TA to go over my test, and I found it was pretty helpful. I realized I didnt have a solid understanding on certain topics, which combined with some silly mistakes, resulted in a poor test grade. It happens. As the semester went on, I participated more in recitation and made sure to ask questions, and at the end of the semester, I actually ended up acing the class. Just goes to show you that you can  finish strong even if you struggle at first. Moral of the story is that MITs a challenging place, but youre never alone if you need help. All you need to do is ask.  Dont be afraid to seek help from your TA, your GRT, or your roommate. Dont forget that office hours and S^3 are a good resource. Most importantly though, dont be afraid to admit that you need help. Its something that a many people here, myself included, struggle with sometimes. But often times, youll save yourself a lot of stress and time if you just talk to someone! Do what you love. I cant stress how important this is. MIT has many activities that you can get yourself involved in. Youll find that here, time will become your most precious commodity. For that reason, find what you really love to do and commit the free time you do have to it. If you really love the activity, youll get a lot more out of it and find an awesome community. On the other hand, if you dont really enjoy it, try something else! Theres lots of choices here, so dont settle for second best. Its all about finding what you like. Plus  with PASS/NR you have no reason to not explore MIT :) Best of luck your first semester! Kirsten

Blind Spots, By Max H. Bazerman And Ann E. Tenbrunsel

When humans hear the term â€Å"blind spots,† they often have a flashback to an event in life where they were driving and attempted to merge into another lane. Typically there are two outcomes from this event: the neighboring driver honks to alert that there is an impending car accident or that accident actually occurs. The same can be said for ethical decisions where humans often do not know they are making a decision with ethical implications. Unfortunately, there is usually not another person to honk at us alerting of the impending danger. The book Blind Spots, by Max H. Bazerman and Ann E. Tenbrunsel, explores these blind spots which pervade ethical decision making for individuals, organizations and society. Throughout the book, the authors offer various areas of day-to-day life where unethical gaps thrive and they offer mechanisms to understand and manage these gaps. Often, unethical human behavior is not intentional, but is coincidentally based on boundaries such as individual knowledge, organizational unanimity, and societal acceptance of policy. On an individual level, although unbeknownst to the individual, humans make decisions based on the best outcomes for themselves, which may result in unintentional and unethical degradation of a fellow human. Further, an organizational setting will compound individual ethical dilemmas as internal groups working together seek acceptance through groupthink, which is the tendency of a work group to come to an agreementShow MoreRelatedThe Ethics Of Blind Spots You Are Given A Lot Different Scenarios Where Both Max H. Bazerman And1057 Words   |  5 PagesWithin Blind Spots you are given a lot different scenarios where both Max H. Bazerman and Anne T. Tenbrunsel test your own ethical values and how you handle them under high pressure situations. What Bazerman and Tenbrunsel are trying to explain and make clear is how we all have blind spots in our ethical point of views. They begin by alerting us of our ethical blind spots so that we are aware of them. They depict the gap between who we want to be and the people we actually are. Anne T. TenbrunselRead MoreStephen P. Robbins Timothy A. Judge (2011) Organizational Behaviour 15th Edition New Jersey: Prentice Hall393164 Words   |  1573 PagesPoint/Counterpoint Employer–Employee Loyalty Is an Outdated Concept 87 Questions for Review 88 Experiential Exercise What Factors Are Most Important to Your Job Satisfaction? 89 Ethical Dilemma Bounty Hunters 89 Case Incident 1 Long Hours, Hundreds of E-Mails, and No Sleep: Does This Sound Like a Satisfying Job? 90 Case Incident 2 Crafting a Better Job 91 4 Emotions and Moods 97 What Are Emotions and Moods? 98 The Basic Emotions 100 †¢ The Basic Moods: Positive and Negative Affect 100 †¢ The

The Music Industry Has Been Revolutionized By...

Over the last fifteen years, the music industry has been revolutionized by technological improvements. It all began with the creation of the mp3 file format; music suddenly became easy to distribute. Napster took advantage of the new file format by creating a peer to peer network, which composed of the first on-demand streaming company. While their company was riddled with lawsuits, they became the initial leaders for digital music. With iTunes’ release, providing users a digital marketplace, the market began its initial shift. The desire to buy and listen to albums’ in entirety diminished; the industry changed to a singles market. In 2008, Spotify noticed the à   la carte downloads that made iTunes quite valuable. Using Napster’s peer to peer model as an initial framework, they created a peer to peer system where they actually paid royalties to the copyright holders. With a freemium service, people could listen to whatever song they wanted as long as they could tolerate intermittent advertisements. Yet what Spotify has managed to do, which no other music streaming company has done, is that they have been able to amass a 40% premium user rate, an astonishing number especially when compared to Pandora’s 5.6% paid user rate. Despite streaming services, particularly Spotify, negative reputation, their entrance into the music industry has not only increased exposure for indie artists, but is also revolutionizing the industry. When Napster was first created, it created buzz likeShow MoreRelatedApple INC analysis1748 Words   |  7 Pagesespecially for its specialization in the personal computers and consumer electronics industry. The company is most well-known for the iPod, a digital music player and Macintosh, a personal computer released in 1984. Co-founded by Steve Jobs in 1976, the company was named under Apple Computers Inc. and its initial product Apple IIe gained relative popularity and success. The release of the Macintosh revolutionized the computer experience with a graphical user interfere and a pointer devise calledRead MoreModern Technology Has Changed Our Lives Essay1696 Words   |  7 Pagesmodern technology from day to day; especially the communication technology has changed the people lives in many ways. Mobile phone is a part of this technology that people can contact each other all over the world through wireless. The invention of this technology has created an unforgettable even in human history, and also the most important for our lives because of its advantages to people in society.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Mobile phone has changed our lives which we can be seen and used every day. People canRead MoreSony Music Entertainment and the Evolution of the Music Industry3835 Words   |  16 PagesSony Music Entertainment and the Evolution of the Music Industry Table of Contents Table of Contents†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦............................2 Recommendations†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦..3 Appendix 1†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 5 Appendix 2†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 11 Appendix 3†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 13 Appendix 4†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 15 Appendix 5†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Read MoreMcdonalds Brand Report Card1364 Words   |  6 Pagesapplies Keller’s brand report card to Apple Inc., brand and evaluates its brand strengths and vulnerabilities against its competitors (Google  ®, Samsung  ®, and Microsoft ®) in efforts to discover areas of weakness and possible opportunities for improvement. 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And like most successful industries, it has experienced drastic developments in technology to ensure growth and success. From the invention of the phonograph in 1877, through the height of vinyl records in the 1970s and the shift to cassettes and compact discs in the eighties, consumers clung to the latest technologies, which made the purchase and ownership of recorded music increasing ly simple. Through each of theseRead Moreâ€Å"from the Mp3 Invention to the Ipod Innovation: the Disruption of the Music Industry†3595 Words   |  15 PagesINTRODUCTION (Confirmation) For the written assignment, I would like to talk to you about the MP3 players industry, at the beginning and more precisely about Apple and the first iPod. Although, Apple doesn’t own the invention of the MP3 format, which was invented in the late eighties, Apple introduced its own MP3 player, the iPod only in 2001and managed to be the firm which popularized the portable music player in the world. 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Athlete Endorsements Free Essays

string(220) " a negative impact on perceptions about a brand, and 4\) Negative publicity generated by the bad behavior of well-known celebrities is harmful because consumers tend to pay more attention to it, then positive publicity\." Sport Marketing and Promotions Introduction What is an athlete endorsement and what significance does it play in the corporate world of advertising? According to dictionary. com an â€Å"endorsement† is the act of endorsing something through approval or sanction. Companies regularly use athletes and other high profile individuals to endorse their company and/or specific products as part of a comprehensive marketing strategy. We will write a custom essay sample on Athlete Endorsements or any similar topic only for you Order Now They use the popularity of their talents to entice consumers to look favorably on their brand and to increase sales of their products or services thru their tacit approvals.These approvals or sanctions are done by having the athlete align themselves with the company thru advertisement, commercials, product or service promotions, or sports gear contract arrangements. Many endorsements deals can bring great wealth and exposure to both an athlete and a company. However, in recent years we have seen the negative side of these endorsement deals when the athlete doesn’t behave in a manner that provides a favorable image to the consumer they wish to attract. This can create difficult decisions for the company when considering whether to use sports athletes to promote their brand and image.The subject of this paper is to explore the Pros and Cons of athlete endorsement arrangements and how they can be either beneficial or detrimental to a company choosing this method of brand awareness. The Origins of Athlete Endorsements In the early 1900’s, Honus Wagner was a well-known professional baseball player who played for the Pittsburgh Pirates. Honus signed a bat contract with Hillerich Bradsby Co. on September 1st, 1905. He was believed to be the first professional athlete with an equipment endorsement deal. During this time period, he was the first major leaguer to have his signature engraved onto a baseball bat.Following Hillerich Bradsby Co’s footsteps was a non-sports company known as Gillette Razor. In 1910, Gillette won the product endorsement deal with major-league baseball stars. Gillette was the 1st non-sport endorsement deal to use professional athletes to market their products (â€Å"History of Athletes,† 2009). With the success of Gillette, more and more companies recognized this great new method of promoting their products and jumped on the proverbial band wagon by hiring high profile professional athletes to market their products.In 1917, with the advent of the Industrial Revolution, Converse All star shoes became the first mass producer of sneakers. With these new production processes, companies began to focus their attention on methods that could attract larger numbers of customers to their products. Converse was quick to recognize the potential that high profile individuals could draw and they became the first to use athlete endorsements in the sneaker industry. Converse used basketball players, such as Akron Firestones and Chuck Taylor to market their shoes (â€Å"History of Athletes,† 2009).It didn’t take long for others to also utilize this new brand awareness that these modern day gladiators could bring to their companies and products and it began a trend many still follow today. However, it wasn’t until the 1960’s when the first issues arose about the negative aspects of athlete endorsements. A prominent Los Angeles attorney, who specialized in the field of athlete endorsements, stated that using an athlete to endorsement a product could be very risky.The example the attorney used to explain th e risk associated with athletes was as follows, â€Å"Suppose a company puts several hundred thousand into a campaign featuring an athlete and suppose that two weeks later the athlete is hurt and not heard from for the rest of the year? † This is something companies have to take into consideration where using athletes to market their products (â€Å"History of Athletes,† 2009). Racial controversies began to rise in the late 1960’s in regards to athlete endorsements.Media sources claimed that black athletes missed out on endorsement deals across the country unless they were playing out of New York, Chicago, or Los Angeles; It’s a subtle form of racism. † In the 1980’s, things changed a lot in regards to using African Americans and athlete endorsements. Michael Jordan’s sneaker, know as Air Jordan’s kicked off in 1986 and became the most successful athlete endorsement in history. Nike sold more than $100 million in a single year, which helped the company hit its first billion-dollar year (â€Å"History of Athletes,† 2009). Athlete endorsements today are seen through a variety of different media ources. You can turn on the television to see Tiger Woods wearing his Nike golf shirt or go online to purchase a football jersey and see Payton Manning using a MasterCard. Athlete’s endorsements have evolved with the latest technologies today and have kept pace in maintaining their relation with their consumers. Companies use Athletes as a marketing strategy to launch new products, reposition products, and/or to reinforce brand images. This paper will explain what researchers have found on the pros and cons of using Athletes to endorse a product. Review of LiteratureIn 2003, a study was conducted to evaluate some of the reasons why celebrity endorsements can be a bad idea for corporate brands. Researchers explained that relying on a single individual, as the exclusive spokesperson for an entire product line may not always be a good idea. Well-known athletes chosen to represent a product in a major advertising campaign can have a huge impact on the company if the athlete behaves in an unprofessional manner. Professional athletes that encounter trouble with the law or act in a manner that is considered culturally undesirable can have a negative ef fect on a company or brand they wish the athlete to endorse. Through this study, researchers came up with a few different conclusions on the reasons why celebrity endorsements can have a negative effect for the companies they represent, 1) Multiple endorsements by the same celebrity can reduce the credibility and increase the liability of the individual celebrity, 2) Because of the increased level of risk associated with using celebrity endorsers, advertisers have begun using more animated characters, 3) Negative information about a celebrity tends to have a negative impact on perceptions about a brand, and 4) Negative publicity generated by the bad behavior of well-known celebrities is harmful because consumers tend to pay more attention to it, then positive publicity. You read "Athlete Endorsements" in category "Papers" If companies want to use celebrities to endorse their products, researchers all agree that the endorser must be both credible and trustworthy.Advertising research indicated that endorsers perceived as being positive role mode ls could also positively influence the perception of the product/brand they are paired endorsing. The study suggests that in order for athletes to be selected for the endorsement job, they should, as a minimum possess credibility and desirable personal characteristics (Stone, Joseph Jones, 2003). Companies pay athletes a lot of money for endorsement deals, so it is the company’s responsibility to do their due diligence when hiring athletes to endorse their brand. Another study done in 2003, identified the key segments of the Canadian sport market and investigated how Generation Y defined a sports hero. The researchers asked several questions to see how this demographic group evaluated heroism. The questions and subsequent answers helped the researchers better understand what types of athletes were selected as heroes, what sport was most frequently associated with heroism, and the important features of heroism. Results reveled that majority of the respondents felt that someone in their family and/or someone who was classified as a sports star was a hero. The sports hero that was selected by the majority of respondents was NBA star Michael Jordan. The participants felt that Jordan’s strength, agility, and celebrity status made him a sports hero. In regards to the sports most frequently associate with heroism, the National Hockey League took 1st place, followed by Major League baseball, National Basketball Association, and National Football League.The results of this study found important implications for sport marketers in regards to the perception of sport heroes and the use of athlete endorsements to target Generation Y customers. As part of the study, they found th at sport endorsements and promotions should emphasize family based promotions, since this generation values family. Sport marketers should also select an athlete who has a positive image since the image carries meaning and relevance to these consumers (Stevens, Lanthrop, Bradish, 2003). A case study was done in 2005 on Soccer Star David Beckham and his athlete endorsements in the international sports industry. Beckham has endorsed several different international companies’ products, such as sports cars, airlines, chocolate, and electronics.Beckham is considered the best-known soccer player in the world, with his personal brand value estimated at $370 million. The case study explained that sponsors consider athletes recognition, appeal to customers, and credibility when choosing an endorser. Since Beckham is a high profile athlete, he is seen as valuable endorser. He is married to Victoria Adams, who is a former female pop star, and continues to be portrayed to the media as a devoted family man. He also uses his fashion to display confidence and sex appeal, which attracts a lot of female fans. The effectiveness of a celebrity endorser, like Beckham himself, relies on physical attractiveness, trustworthiness, personal characteristics, and cultural meaning.Companies, such as Gillette have used Beckham in their marketing strategy to target a younger, global consumer. Gillette feels that Beckham could improve their image and boost its brand internationally. By using Beckham, they can reach men aged 18-34. Findings have indicated that customers have a more positive brand attitude and purchasing intentions when the product is paired with an attractive celebrity endorser. Since David Beckham is considered a high profile athlete with physical attractiveness, he is a good fit for endorsements. The conclusions from this case study indicate both sports organizations and corporations use elite athletes to expand their fan base and reach their global target markets successfully.Sports celebrities are appealing to global market players and can be used in other sport marketing activities, such as venue naming rights or event sponsorships. Given the advances in technology today, the growing interest in sports marketing is becoming more global then ever and future studies should be conducted to see what marketing strategies are most effective (Yu, 2005). David Beckham is a good example of an effective Athlete endorser who was used on an international level. In July of 2007, research was conducted on the effects of athlete endorsements on attitudes towards the product. Companies use athlete endorsements as a promotional strategy in launching new products, repositioning brands, or reinforcing brand images.In some endorsement strategies, athletes can be seen advertising non-sport pro ducts, such as computer products or electronic equipment. Marketing research asks the question, â€Å"Do consumers react favorably to non-sport endorsed products? † This study found that endorsers are more effective when there is a corresponding relationship between the endorser and the endorsed product. Researchers looked at the compatibility, creditability, attractiveness, and attitudes of individuals towards the athlete endorser and product they advertise (Kim Na, 2007). The analysis revealed that when an athlete endorses sports products, such as running shoes the credibility and attractiveness are more favorable to the consumer than non-sports products, such as perfume.Consumers see more of a relationship between the athlete and the product, especially if it related to the sport in which the athlete participates. For example, Tiger Woods can be seen wearing a Nike golf shirt when playing in the PGA Tour. This helps to show Tiger’s compatibility and credibility with the Nike brand. In 2008, Brad Carlson and Todd Donavan co nducted a study to look at athlete endorser’s effect on both brand and team-related outcomes. The study applied a social identity framework to investigate consumer responses to athlete endorsements. Researchers looked at the cognitive state of self-categorization, which exists when a fan feels belongingness with an entity, such as an athlete.They also looked at athlete identification, which is a cognitive state in which the individual evaluates the degree of overlap between their own self-schema and the athlete’s schema. The results found that fans that identified more strongly with an athlete were more likely to purchase the endorsed products. Athletes are effective endorsers when fans aspire to be like them, for example a person may associate himself or herself with the player by wearing their jersey. Fans who were identified less with an athlete endorser were more likely to abandon the teams market offerings. The findings confirmed that identifying with an athlete endorser is an important determinant of both team and brand related outcomes (Carison Donavan, 2008). Athlete Endorsements There are both pros and cons of athlete endorsements.Using an athlete as a spokesperson for an organization can be highly effective tool in building brand awareness, but it depends mostly on the target audience and how they relate to the athlete. According to Brooks International Online, some of the benefits of using an athlete to endorse products consist of raising awareness, generating media coverage, and attracting new audiences (Stuart, 2009). When selecting an athlete, companies should pick an individual whose demographics appeal to the target audience. Companies should also pick an athlete who will add value to the product they are endorsing. This past year, a case study was done on the marketing actives of Under Armor. The case looked at what types of marketing activities they used to help compete with their competitors. The main focus of this case was on athlete endorsements.Since the owner of Under Armor was an athlete himself, he felt the best way to advertise his product was by the use of athletes. He said that athletes are like a walking billboard and they help increase word of mouth marketing in the workplace (Kraft Lee, 2009). According to this article, in today’s marketplace, athlete endorsers are used to capture of attention of consumers, strengthen recall of the brand name, reinforce brand image, give the message credibility, increase product attractiveness, and increase the likelihood of purchase. The conclusion from this study showed that using athletes to endorse their product brought on more media attention then any other promotional strategies.The remaining section of this paper will give some examples of the pros and cons of athlete endorsements that have taken place over the past 20 years. PROS The following are just a few examples of positive athlete endorsers; Dale Earnhard t, Michael Jordan, Tiger Woods, LeBron James, and the Williams Sisters. Dale Earnhardt, who was teamed up with Pepsi, helped generate awareness for the energy drink Amp Energy. Pepsi feels that attaching an athlete to the marketing of their product is crucial for brand awareness. The endorsement deal with Earnhardt consisted of the Pepsi logo on his racecar and on his racing gear. When Earnhardt made the front cover of Sports Illustrated Magazine, this was huge for Pepsi.Earnhardt was basically a walking billboard for Pepsi, everywhere he was seen, and so was Pepsi (â€Å"Pepsi makes heavy,† 2009). The most successful and well-known athlete endorsement in history was Michael Jordan and Nike. The slogan that was created during Jordan’s endorsement with Nike was â€Å"Be like Mike. † Michael Jordan’s sneaker, know as Air Jordan’s kicked off in 1986 and Nike sold more than $100 million in a single year (â€Å"History of Athletes,† 2009). Michael Jordan created brand awareness and loyalty for the Company, which in return made Nike one of the best known show companies in the world. Not much longer after Jordan, Nike picked up another star athlete known as Tiger Woods. Tiger is the highest paid Athlete endorser at this time. According to Forbes. om, Tiger is the world’s top golfer and made nearly $110 million in endorsements from June 2008 to June 2009. Some of the other endorsement deals Tiger has are Buick, Gatorade, Tag Huger, ATT and Gillette. Gatorade launched Tiger Gatorade in March of 2008, which claims that it â€Å"helps focus your mind and your body†. Tiger actually picked out the flavor himself and even gets a percentage of all sales sold under his name. Tiger is predicted to be the number one sports endorser again in 2010 (Paulson, 2007). Another great sports endorser is Lebron James. Lebron James entered into the NBA right after High School at age 18. The same day James signed with the Cleveland Cavilers, Nike signed him for a $90 million endorsement deal (â€Å"Pros and Cons†, 2009).This deal was another big hit for Nike. LeBron and the Cavilers have been a playoff team for several years now and Nike has enhanced their image by using athletes who have a history of winning. The final example of positive athlete endorsements is the Williams Sisters. Both Serena and Venus have endorsement deal with large companies due to their high profile status in professional Tennis. Serena Williams currently has deals with Nike, Kraft, Wilson, and Hewlett Packard (â€Å"Serena williams hopes,† 2009). Serena just singed a new deal with Tampax. After Serena’s outburst at the line judge in the 2009 US Open, tampax decided to keep Serena as the endorser and use her temper in the ads.His latest advertisement shows Serena taking her temper out on a woman’s dressed as Mother Nature (Picchi, 2009). Serena’s older sister Venus Williams signed the most lucrative deal ever for a female athlete with Reebok. Both Sisters’ have been classified as positive athlete endorses and help build brand awareness for the companies they endorse. CONS The following are some examples of negative athlete endorsers; Michael Vick, Kobe Bryant, Manny Ramirez, and Michael Phelps. Michael Vick, who was convicted of dog fighting crimes in 2007, lost his endorsements deals with several different companies. Nike who was just about to launch the Air Zoon Vick V shoe cut all endorsement ties with Vick immediately after his conviction (Lippert, 2007).They removed all items bearing Vick’s name from company stores. This hit Nike hard financially. Reebok also pulled all Vicks jerseys and merchandise from their shelves. AirTran Airways stripped Vicks contract and made him give back his $20-$35 million signing bonus money (Kruse, 2009). All of the companies who endorsed Vick felt the negative impact on their companies. The second example is Kobe Bryant. Bryant who endorsed companies such as Nike, McDonald’s, Sprite, and Rawlings was convicted for sexually assaulting a 19-year-old woman in 2003. McDonald’s got hit the worst from this outburst since its marketing strategies are geared towards mothers and families.The study by ESPN found that incidents such as Bryant’s tarnish a company’s image (Rovell, 2003). Regardless of whether or not Bryant sexually assaulted this woman, the companies associated with him felt a negative impact through using him to promote their products. Another athlete who had a negative impact on an endorsement deal was Manny Ramirez. Ramirez, who plays for the LA Dodgers used performance-enhancing drugs and got suspended for 50 games (Jones, 2009). The game company EA Sports used Ramirez to endorse their products. Since EA sports targets younger teenagers, this had a negative impact on their brand. What mother would want to purchase a video game with Ramirez on the cover?These are some of the issues EA Sports now faces with their image. The final example of a negative athlete endorsement is Michael Phelps and his endorsement deal with Kellogg’s. Michael Phelps who is an Olympic gold medalist, had some issues with the law with drinking and driving. He was also found smoking marijuana from a picture that was posted on the Internet. Even though some companies stuck by him, Kellogg’s dropped Phelps immediately (McCarthy, 2009). Kellogg’s main target market is mothers and children. This puts a bad image on Kellogg’s to endorse an athlete who drinks and takes drugs. Ever since Phelps issue with the law, companies now see him as a risk. ConclusionThere are both pros and cons with using athletes for endorsements. Companies use Athletes as a marketing strategy to launch new products, reposition products, and/or to reinforce brand images. In many cases, the affiliation is a positive one that brings great financial reward to both an athlete and the company. When companies select athletes of high integrity and character, they impart a positive brand image on their products and generally reap the rewards of increased sales and revenue. However, when companies hire endorsers who do things culturally unacceptable for the products they are trying to sell, the relationship can have a negative impact on the bottom line.This has caused some concern among company executives and many are questioning the use of athletes to promote their products due to the risk associated with not being able to control their actions on and off their field (Sandomir, 2007). Researchers question how credible and believable athlete endorsements are in promoting their products but some case studies have proven that using athle tes to endorse sports products is more effective then endorsing non-sports products. This allows the consumer to see a relationship between the endorser and the product being endorsed. According to Millsport. com, companies need to pick an athlete who they can trust, has credibility, and demonstrates moral character.Avoiding risky endorsement deals with questionable athletes is a primary concern to decision maker’s at large national and international companies. It is important, if using an athlete to endorse your product, that a company does their due diligence in investigating the high profile individual and that it makes economic sense to insure a positive return on the investment. The athlete must fit their marketing strategies and fit the target market they are trying to reach. If companies do find an athlete that provides a good fit with their organization and brand, then using an athlete to endorse their products could be a successful way of raising brand awareness and revenue.However, do your homework as a bad endorsement deal can quickly tarnish a brands image that has taken many years to build. There are many endorsement opportunities that can build additional sales but companies need to be aware of the aspects that could negatively affect their business. References (2009). Millsport and the marketing arm. Retrieved from http://millsport. com (2009, September). History of athlete endorsements. Retrieved from http://google.com (2009). Serena williams hopes to ride u. s. open to win new endorsements. Sports Business Daily, Retrieved from http://www.sportsbusinessdaily. com/article/123828 (2009, September 5). How to cite Athlete Endorsements, Papers

Real Time Pcr free essay sample

PROBE-BASED DETECTION SYSTEMS14 Hybridization probes (also called FRET probes)16 MELTING CURVE ANALYSIS16 Multiplex real-time PCR18 APPLICATIONS OF REAL TIME PCR18 GENE EXPRESSION ANALYSIS18 SNP GENOTYPING19 HIV DETECTION19 CYSTIC FIBROSIS (CF) DETECTION:20 THE ADVANTAGES OF REAL-TIME PCR20 THE DISADVANTAGES21 REFRENCES21 REAL TIME PCR TRADITIONAL PCR The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be copied, or â€Å"amplified†, many thousand- to a millionfold. In traditional (endpoint) PCR, detection and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis. REAL-TIME QUANTITATIVE PCR (qPCR) In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision. WHAT’S WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. Short dynamic range lt; 2 logs. * Low resolution. * Non-automated. * Size-based discrimination only * Ethidium bromide staining is not very quantitative REAL TIME PCR VS PCR . BASIC PRINCIPLE Quantitative PCR  is carried out in a  thermal cycler  with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited  fluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of the  nucleic acids  and  DNA polymerase. The PCR process generally consists of a series of temperature changes that are repeated 25 – 40 times, these cycles normally consist of three stages: the first, at around 95  Ã‚ °C, allows the separation of the nucleic acid’s double chain; the second, at a temperature of around 50-60  Ã‚ °C, allows the alignment of the primers with the DNA template;  the third at between 68 72  Ã‚ °C, facilitates the  polymerization  carried out by the DNA polymerase In real-time PCR, * the amount of DNA is measured after each cycle by the use of fluorescent markers that are incorporated into the PCR product. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. * Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. * The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1). Figure 1—Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples being amplified in this example are a dilution series of the template. TYPES OF PCR Quantitative PCR| Qualitative qPCR| A specific or non-specific detection chemistry allows the quantification ofthe amplified product. | In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. | The amount detected at a certain point of the run is directly related to theinitial amount of target in the sample| For virus sub-typing and bacterial species identification. Can also be used for allelic discrimination between wild type and mutant, between different SNPs or between different splicing forms. | common pplications of quantitative PCR are gene expression analysis, pathogen detection/quantification and microRNA quantification| Different fluorophores can be used for the two alleles, and the ratio of the fluorophores signals correlates to the related amount of one form compared to the other one. | Quantitative PCR software uses the exponential phase of PCR for quantification. | Specific detection methods such as Double-Dye probe systems are more ofte n used for theseApplications| Overview of real-time PCR Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined.. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real time PCR instruments that measure fluorescence while performing temperature changes needed for the PCR cycles. qPCR STEPS There are three major steps that make up a qPCR reaction. Reactions are generally run for 40 cycles. 1. Denaturation—The temperature should be appropriate to the polymerase chosen (usually 95 °C). The denaturation time can be increased if template GC content is high. 2. Annealing—Use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5 °C below the Tm of the primer). 3. Extension—At 70–72 °C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in qPCR is small, this step is often combined with the annealing step using 60 °C as the temperature. BASICS OF REAL TIME PCR Baseline – The baseline phase contains all the amplification that is below the level of detection of the real time instrument. Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically CT – (cycle threshold) the cycle number where the fluorescence passes the threshold Rn – (Rn-baseline) NTC – no template control Rn is plotted against cycle numbers to produce the amplification curves and gives the CT value. ONE-STEP OR TWO-STEP REACTION qRT-PCR can be one step or two step. 1. Two-step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase (RT). This first-strand cDNA synthesis reaction can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs). To give an equal representation of all targets in real-time PCR applications and to avoid the 3? bias of oligo(dT), it is usually recommended that random hexamers or a mixture of oligo(dT) and random hexamers are used. The temperature used for cDNA synthesis depends on the RT enzyme chosen. Following the first-strand synthesis reaction, the cDNA is transferred to a separate tube for the qPCR reaction. In general, only 10% of the first strand reaction is used for each qPCR. . One-step qRT-PCR One-step qRT-PCR combines the first-strand cDNA synthesis reaction and qPCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specifi c primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspecific products in the one-step procedure and reduce the amount of product of interest. O verview of qPCR and qRT-PCR components This section provides an overview of the major reaction components and parameters involved in real-time PCR experiments. * DNA polymerase One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA, allowing the polymerase to synthesize nonspecific product. The problem of nonspecific products resulting from mispriming can be minimized by using a â€Å"hot-start† enzyme. Using a hot-start enzyme ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. * Template Anywhere from 10 to 1,000 copies of template nucleic acid should be used for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 ? of genomic DNA, or cDNA, generated from 1 pg to 100 ng of total RNA. Excess template may increase the amount of contaminants and reduce efficiency. If the template is RNA, care should be taken to reduce the chance of genomic DNA contamination. One option is to treat the template with DNaseI. Ultrapure, intact RNA is essential for full-length, high-quality cDNA synthesis and accurate mRNA quantification. RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. * Reverse transcriptase The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase. It is important to choose an RT that not only provides high yields of full-length cDNA but also has good activity at high temperatures. High-temperature performance is also very important for tackling RNA with secondary structure or when working with gene-specific primers (GSPs). * dNTPs It is recommended that both the dNTPs and the Taq DNA polymerase be purchased from the same vendor, as it is not uncommon to see shifts of one full threshold cycle (Ct) in experiments that employ these items from separate vendors. * Magnesium concentration In qPCR, magnesium chloride or magnesium sulfate is typically used at a fi nal concentration of 3 mM. This concentration works well for most targets; however, the optimal magnesium concentration may vary between 3 and 6 mM. * UNG The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every run it is possible to get rid of any previous PCR product. * ROX Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines. If you work with such machines, it is easier to work with the ROX dye already incorporated in the MasterMix rather than adding it manually. It guarantees a higher level of reproducibility and homogeneity of your assays. * Fluorescein For iCycler iQ, My iQ and iQ5 machines (BioRad thermocyclers), the normalization method for SYBR Green assay uses Fluorescein to create a â€Å"virtual background†. As in the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays. REAL TIME PCR SYSTEM: System Features: †¢ Four interchangeable block formats †¢ Optional Automation Accessory amp; Barcode Scanner †¢ Argon ion laser/CCD camera †¢ Easy to Use Software, Multiple Applications †¢ Set up Wizards †¢ QC Filtering/Flag System †¢ Flexible data reports amp; exporting SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 1 ) Light Cycler ® Relative Quantification Software The first commercially available software was the Light Cycler ® Relative Quantification Software (2001). 2 ) REST In 2002, the relative expression software tool (REST ) was established as a new tool. 3 ) Q-Gene Recently a second software tool, Q-Gene, was developed, which is able to perform a statistical test of the real-time data. Q-Gene manages and expedites the planning, performance and evaluation of quantitative real-time PCR experiments. 4) OligoPerfect A primer design software program such as OligoPerfectâ„ ¢, available on the Web at www. invitrogen. com/oligoperfect, can automatically evaluate a target sequence and design primers for it based on the criteria STEPS OF REAL TIME PCR Real-time reaction mix (final concentrations): 1x 2 x AmpliTaq Gold 0. 5 ? M 5’ primer 0. 5 ? M 3’ primer 0. 2 ? M probe 0. 4 ? Rox reference dye 20 ? l Final Volume (including sample and dH20) STANDARD REAL-TIME PCR PROTOCOL ASSAY DESIGN: This section describes the stages of real-time PCR assay design and implementation. We will identify sources of variability, the role they play in data accuracy, and guidelines for optimization in the following areas: 1Target amplicon and primer design 2. Nucleic acid purification 3. Reverse transcription 4. Controls and normalization 5. Standard curve evaluation of efficiency, sensitivity, and reproducibility Good primer (pair) properties One way to minimize efficiency bias is to amplify relatively short targets. Amplifying a 100 bp region is more likely to result in complete synthesis in a given cycle than, say, amplifying a 1,200 bp target. For this reason, real-time PCR target lengths are generally in the range of 60 bp to 200 bp. In addition, shorter amplicons act as a buff er against variations in template integrity. Primers designed to amplify larger regions are less likely to anneal with the same fragment in a slightly degraded nucleic acid sample. PURIFICATION Phenol-based organic extraction is a very effective method for purifying RNA from a wide variety of cell and tissue types. During sample lysis, phenol and guanidine isothiocyanate disrupt cells and dissolve cell components. while maintaining the integrity of the nucleic acids by protecting them from RNases. Chloroform is added and the mixture is separated by centrifugation, which separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase in the presence of guanidine isothiocyanate, while DNA and protein are driven into the organic phase and interphase. The RNA is then recovered from the aqueous phase by precipitation with isopropyl alcohol. REVERSE TRANSCRIPTION CONSIDERATIONS Most reverse transcriptases employed in qRT-PCR are derived from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M-MLV). An ideal reverse transcriptase will exhibit the following attributes: * Thermostability— thermostable RTs function at the higher end of (or above) this range and allow for successful reverse transcription of GC-rich regions. * RNase H activity— RNase H activity can drastically reduce the yield and ratio of full-length cDNA, which translates to poor sensitivity. Several RTs, most notably SuperScript II and III, have been engineered for reduced RNase H activity. NORMALIZATION AND QUANTIFICATION: When analyzing and comparing results of Real-Time qPCR assays many researchers are confronted with several uncontrolled variables, which can lead to misinterpretation of the results. Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and differences between tissues, individuals or experimental conditions. In order to make a good comparison, normalization can be used as a correction method, for these variables. The most commonly known and used ways of normalization are : * normalization to the original number of cells, * normalization to the total RNA mass, normalization to one or more housekeeping genes, * normalization to an internal or external calibrator. Normalization to number of cells can actually only be done for cell culture and blood samples. The two majors methods of normalization are the absolute quantification and the relative quantification . Absolute quantification Absolute quantification requires a standard curve of known copy numbers. The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can be used. The standard must be amplified using the same primers as the gene of interest and must amplify with the same efficiency. The standards must also be quantified accurately. This can be carried out by reading the absorbance at A260, although this does not distinguish between DNA and RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. Relative quantification Relative quantification is the most widely used technique. Gene expression levels are calculated by the ratio between the amount of target gene and an endogenous reference gene, which is present in all samples. The reference gene has to be chosen so that its expression does not change under the experimental conditions or between different tissue. There are simple and more complex methods for relative quantification, depending on the PCR efficiency, and the number of reference genes used. STANDARD CURVE TO ASSESS EFFICIENCY, SENSITIVITY, AND REPRODUCIBILITY The final stage before assay employment is validating that all the experimental design parameters result in a highly efficient, sensitive, and reproducible experiment. * Reaction efficiency One hundred percent efficiency corresponds to a perfect doubling of template at every cycle, but the acceptable range is 90–110% for assay validation. This efficiency range corresponds to standard curve slopes of –3. 6 to –3. 1. The graph in Figure shows the measurement bias resulting solely from differences in reaction efficiency.. A standard curve is generated by plotting a dilution series of template against the Ct for each dilution. To some, sensitivity is measured by how early a target Ct appears in the amplification plot. However, the true gauge of sensitivity of an assay is whether a given low amount of template fits to the standard curve while maintaining a desirable efficiency. The most dilute sample that fits determines reaction sensitivity. The standard curve also includes an R2 value, which is a measure of replicate reproducibility. Standard curves may be repeated over time to assess whether the consistency, and therefore the data accuracy for the samples. Real-Time PCR Fluorescence Detection Systems Several different fluorescence detection technologies can be used for realtime PCR, and each has specific assay design requirements. All are based on the generation of a fluorescent signal that is proportional to the amount of PCR product formed. The three main fluorescence detection systems are: * DNA-binding agents (e. g. SYBR Green and SYBR GreenER technologies * Fluorescent primers (e. g. , LUX Fluorogenic Primers and Amplifluor qPCR primers) * Fluorescent probes (e. g. , TaqMan probes, Scorpions, Molecular Beacons) The detection method plays a critical role in the success of real-time PCR. DNA-Binding Dyes The most common system for detection of amplified DNA is the use of intercalating dyes that fluoresce when bound to dsDNA. SYBR Green I and SYBR GreenER technologies use this type of detection method. The fluorescence of DNA-binding dyes significantly increases when bound to double-stranded DNA (dsDNA). The intensity of the fluorescent signal depends on the amount of dsDNA that is present. As dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detected using real-time PCR instruments. SYBR Green I advantages †¢ Low cost assay †¢ Easy design and set up SYBR Green I disadvantages †¢ Non specific system †¢ Not adapted to multiplex †¢ Non suitable for qualitative qPCR Primer-Based Detection Systems Primer-based fluorescence detection technologies can provide highly sensitive and specific detection of DNA and RNA. In these systems, the fluorophores is attached to a target-specific PCR primer that increases in fluorescence when incorporated into the PCR product during amplification. * Amplifluor Real-Time PCR Primers Amplifluor real-time PCR primers are designed with both a fluorophore and quencher on the same primer. The primer adopts a hairpin configuration that brings the fluorophore in close proximity to the quencher. The fluorescent signal increases when the primer is unfolded and the fluorophore and quencher are de-coupled during incorporation into an amplification product. Figure: Ampliflour primer PROBE-BASED DETECTION SYSTEMS Probe-based systems provide highly sensitive and specifi c detection of DNA and RNA. However, dual-labeling and complex design specifi cations make them expensive and more diffi cult to use than primer-based systems or DNAbinding dyes. TaqMan probes = Double-Dye probes TaqMan probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual Labelled probes, are the most widely used type of probes. A fluorophore is attached to the 5’ end of the probe and a quencher to the 3’ end. The fluorophores is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy Transfer) to the quencher. TaqMan probes can be used for both quantification and mutation detection, and most designs appear to work well. TaqMan ASSAY DENATURATION ANNEALING OF PRIMERS AND PROBE POLYMERIZATION AND PROBE CLEAVAGE Molecular Beacons In addition to two sequence-specific primers, molecular beacon assays employ a sequence-specific, fluorescently labeled oligonucleotide probe called a molecular beacon, which is a dye-labeled oligonucleotide (25–40 nt) that forms a hairpin structure with a stem and a loop . A fluorescent reporter is attached to the 5 end of the molecular beacon and a quencher is attached to the 3 end. The loop is designed to hybridize specifically to a 15–30 nucleotide section of the target sequence Figure: Moleculer Beacon They are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur a desirable quality for allelic discrimination experiments. Hybridization probes (also called FRET probes) Roche has developed hybridization probes for use with their LightCycler. Two probes are designed to bind adjacent to one another on the amplicon. One has a 3’ label of FAM, whilst the other has a 5’ LC dye, LC red 640 or 705. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes hybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. FRET probe principle and light cycler MELTING CURVE ANALYSIS Melting curve analysis can only be performed with real-time PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR Green I or SYBR GreenER dye primers can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan probes are not compatible because they produce an irreversible change in signal by cleaving and releasing the fluorophore into solution during the PCR; however, the increased specificity of this method makes this less of a concern. The level of fluorescence of both SYBR Green I and SYBR GreenER dyes significantly increases upon binding to dsDNA. By monitoring the dsDNA as it melts, a decrease in fluorescence will be seen as soon as the DNA becomes single-stranded and the dye dissociates from the DNA. Figure: Melting curve analysis can detect the presence of nonspecifc products, as shown by the additional peaks to the left of the peak for the amplified product in the melt curve. How to perform melting curve analysis To perform melting curve analysis, the real-time PCR instrument can be programmed to include a melting profile immediately following the thermocycling protocol. After amplification is complete, the instrument will reheat your amplified products to give complete melting curve data. Most real-time PCR instrument platforms now incorporate this feature into their analysis packages. In general, the program steps will be: 1. Rapid heating of the amplified sample to 94 °C to denature the DNA. 2. Cooling the sample to 60 °C. 3. Slowly heating (by increasing the temperature 0. 2 °C/second) the sample while plotting fluorescence signal vs. temperature. (As the temperature increases and the dsDNA strands melt, the fluorescence signal will decrease. ) Figure: Example of a melting curve thermal profile setup on an Applied Biosystems instrument (rapid heating to 94 °C to denature the DNA, followed by cooling to 60 °C. ) Multiplex real-time PCR In multiplex real-time PCR, more than one set of gene-specific primers is used to amplify separate genes from the template DNA or RNA in a single tube. Typically, multiplex reactions are used to amplify a gene of interest and a â€Å"housekeeping† gene (e. g. , #-actin or GAPDH), which is used as a normalize for the reaction. Because more than one PCR product will be quantified in the same tube, different fluorescent reporter dyes are used to label the separate primers or probes for each gene. More Samples Analyzed per Plate. Target and normalizer in same reaction and Less sample consumed. APPLICATIONS OF REAL TIME PCR GENE EXPRESSION ANALYSIS A sample gene expression analysis using a multiplex TaqMan assay is presented in the following sections. In this example, we’re interested in the relative expression of three genes in the polyamine biosynthesis pathway, ornithine decarboxylase (ODC), ODC antizyme (OAZ), and antizyme inhibitor (AZI), in two different samples, sample A and sample B. 1. RNA was isolated from sample A and sample B. 2. RNA was reverse transcribed into cDNA. 3. The amount of the target genes (ODC, OAZ, and AZI) and the reference gene (b-actin) was determined in each of the cDNA samples using a multiplex qPCR assay. 4. Data were analyzed and the relative expression of each of the target genes in the two samples was calculated. EXAMPLE BRCA1 is a gene involved in tumor suppression. BRCA1 controls the expression of other genes. In order to monitor level of expression of BRCA1, real-time PCR is used. SNP GENOTYPING In order to perform SNP genotyping, two specific probes labeled with different dyes are used, the first for the wild type allele and the second for the mutant allele. If the assay results in the generation of only the first fluorescent color, then the individual is homozygous wild type at that locus. If the assay results in the generation of only the second fluorescent color, then the individual is homozygous mutant. And finally, if both fluorescent colors are produced, then the individual is heterozygous. At the end of the reaction, hydrolysis probes are digested. The quality of a hydrolysis probe is given by the hybridization efficiency, the quenching of the intact probe and the cleavage activity of Taq polymerase. HIV DETECTION Nowadays HIV is strikingly spreading out whole the world. so in order to diminish its distribution , it is necessary to detect it as soon as possible amp; for this purpose, Real time PCR is recommended by scientist. In this method ,’ pol’’ gen of the virus, is amplified in thermocycler. 6 patient have been studied. infection in these patients was confirmed by ELISA amp; western blot. * Sampling amp; RNA extracting from patients. * Cloning of target segment by using Xba I amp; Hind III. And 180 bp primers. * Standard virus mRNA was extracted. * Quantitative analysis of HIV virus by SYBR-green Real Time RT-PCR. CYSTIC FIBROSIS (CF) DETECTION: Cystic f ibrosis (CF) is the most common inherited disease among Caucasian populations with an incidence of ~1 in 2500 births. A3 base pair (bp) deletion, designated DF508, accounts for nearly 70% of CF cases and causes severe manifestations of the disease. It results in the absence of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and this error prevents normal processing and translocation of the polypeptide chain to apical membranes of epithelial cells. This deletion can be detected by molecular beacons in real time PCR. Figure:Examples of specific molecular beacon fluorescence increase during real-time PCR in samples containing single lymphoblasts homozygous normal for CF (green), heterozygous DF508 (blue), or homozygous DF508 (red). A) Fluorescent signal from the molecular beacon detecting the normal allele. (B) Fluorescent signal from the molecular beacon detecting the DF508 allele. Dashed lines indicate the threshold of 200 units (~10 SD above baseline readings) used for determining CT values. THE ADVANTAGES OF REAL-TIME PCR * The ability to monitor the progress of the PCR reaction as it occurs in real time * The ability to precisely measure the amount of amplicon at each cy cle * An increased dynamic range of detection * The combination of amplification and detection in a single tube, which eliminates post-PCR manipulations. Rapid cycling times (1 hour) * High sample throughput (~200 samples/day) * Low contamination risk (sealed reactions) * Very sensitive (3pg or 1 genome eq of DNA) * Broad dynamic range (10 1010 copies) * Reproducible (CV lt; 2. 0 %) * Allows for quantitation of results * Software driven operation * No more expensive than â€Å"in house† PCR ($15/test) THE DISADVANTAGES * Current technology has limited capacity for multiplexing. Simultaneous detection of 2 targets is the limit. * Development of protocols needs high level of technical skill and/or support. Requires Ramp;D capacity and capital) * High capital equipment costs ($ 50,000 -160,000). REFRENCES * http://www. icmb. utexas. edu/core/DNA/qPCR/QiagenRT-PCR. pdf www. icmb. utexas. edu * http://books. google. com. pk/books? id=-v-U-mXWg-gCamp;printsec=frontcoveramp;dq=real+time+pcramp;hl=enamp;sa=Xamp;ei=Bph1UezKIceDhQeUh4CwCAamp;ved=0CDAQ6AEwAQ#v=onepageamp;q=real%20time%20pcramp;f=false books. google. com. pk * PCR/Real-Ti me PCR Protocols www. protocol-online. org Real-Time Pcr: An Essential Guide Google Books books. google. com * * http://www. gene-quantification. e/bio-rad-CFX96-bulletin-5589. pdf www. gene-quantification. de * https://www. google. com. pk/#output=searchamp;sclient=psy-abamp;q=fret+rt-qpcramp;oq=fret+in+rtamp;gs_l=serp. 1. 1. 0i22i30l2. 1583. 4622. 1. 10196. 6. 6. 0. 0. 0. 0. 551. 2584. 3-3j1j2. 6. 0 0. 0 1c. 1. 9. serp. 97Wjtm9UCU4amp;psj=1amp;bav=on. 2,or. r_cp. r_qf. amp;fp=f6d28cf5fd703914amp;biw=1366amp;bih=600 www. google. com. pk * BioTechniques Real-time PCR for mRNA quantitation www. biotechniques. com * http://env1. gist. ac. kr/joint_unugist/file/g_class11_real_time_pcr_vt. pdf env1. gist. ac. kr