The Polymerase Chain Reaction Pcr

The Polymerase Chain Reaction Pcr The polymerase chain reaction was first developed in 1983 by Kary Mullis. This reaction is commonly used in molecular biology to amplify and generate thousands to millions of copies of specific DNA sequences across several orders of magnitude (4-1). It relies on thermal cycling, consisting of cycles of denaturation, primer (short DNA fragment) annealing and primer extension (4-7). PCR can also be used for the analysis of RNA sequences and to qualitatively detect RNA expression levels through creation of complementary DNA (cDNA) transcripts from RNA by use of reverse transcriptase. This technique is called reverse transcription-PCR (RT-PCR) (5-2). Although PCR and RT-PCR have revolutionized many areas of biomedical science, they are not suitable for the quantitative analysis of analysis of samples. Hence, real-time or quantitative PCR (qPCR) techniques need to be employed (5-8, 5-9). RT- qPCR distinguishes itself from other methods available for gene expression, such as northern-blot analysis, ribonuclease (RNase) protection assay and competitive RT-PCR, in term of accuracy, sensitivity and fast results (2,6). RT-qPCR does not required post-amplification manipulation and it can produce quantitative data with wide dynamic range of detection (7 to 8 logs). In addition, RT-qPCR assay is 10,000 to 100,000-fold more sensitive than RNase protection assays and 1000-fold more sensitive than dot blot hybridization (3). RT- qPCR also can even detect a single copy of a specific transcript and can reliably detect gene expression differences as small as 23% between samples (3-6, 3-7). Furthermore, it has lower coefficients variation (cv; TaqMan at 24%; SYBR Green at 14.2%) than end point assays such as probe hybridization and band densitometry (45.1%; 44.9% respectively) (3-8). RT- qPCR can differentiate between messenger RNAs (mRNAs) with almost identical sequences and requires much less RNA template than other methods of gene expression analysis. Because of this, RT- qPCR has established itself as the gold standard for the detection and quantification of RNA targets (1-2,2). Furthermore, it is firmly established as a mainstream research technology (1-3). However, the major disadvantage of RT-qPCR is that required expensive equipment and reagents (3). The principle of RT-qPCR is straight forward: following the reverse transcription of RNA in to cDNA, it needs an appropriate detection chemistry to dete ct the presence of PCR products, an instrument to monitor the amplification in real-time and compatible software for quantitative analysis. RT- qPCR is characterized by the point in time during cycling when a PCR product amplification is first detected (Figure 1, 1). A direct relationship between the starting copy number of the nucleic acid target and the time required to observe fluorosence increasing. Nowadays, there are four fluorescent DNA probes available for RT-qPCR detection of PCR products: TaqMan, SYBR Green, Molecular Beacons, and Scorpions. All of them generate a florescent signal to allow the detection of PCR products. While the TaqMan probes, SYBR Green, Molecular Beacons, and Scorpions generation of fluorescence depend on Forster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates, the SYBR Green dye simply emits its fluorescent signal by binding to the double-strand DNA in solution (5-34). As RT-qPCR has extremely high sensitivity and reproducibility, in depth understanding of normalization techniques is imperative for accurate conclusions (6). Normalization of gene expression data is an essential component of a reliable RT-qPCR assay and it is used to control for error between samples (7,3). This error could be introduced at one or more stages throughout the experimental protocol; (input sample, RNA extraction, etc.) however, there are many strategies to control this error ( please, read the discussion section strategies for more details). Currently, internal control genes, which are often referred to as housekeeping genes, are most frequently used to normalize the messenger RNA (mRNA) fraction. This housekeeping gene should remain constant in the tissues on cells under investigation, or in response to experimental treatment (8, 8-69, 8-70, 6,7). In addition, the ideal housekeeping genes should by stably expressed, and their abundances should show strong correlation w ith mRNA total amounts present in the samples (8). Consequently, normalization against a single housekeeping gene in not acceptable and can falsely bias results unless the researchers present clear evidence for the reviewers that confirms its invariant expressions conditions described (3,8-71,8-73). In this study we carried out an evaluation the gene expression of three commonly used housekeeping genes (GAPDH, ÃŽÂ ²-action, ALAS1) in three different cell lines which are derived from T-cell leukemia, B-cell lymphoma and myeloid leukemia, using RT-qPCR as an analytical tool. Our goal was to recognize a housekeeping gene with minimal variability under different experimental conditions. Materials and Methods: Samples, RNA handling and isolation: Cell line pellets (5-10X106 cells) which have been frozen in 0.5 ml TRIsure Reagent (Bioline code BIO-38033). Cell lines are: Jurkat, CEM-C7 and MOLT-4 (all T-cell leukemia-derived); SKW 6.4, BJAB and JeKo-1 (all B-cell lymphoma-derived); and HL-60, NB4 and K562 (all derived from myeloid leukemia). RNA was isolated from cell pellets using Trizol procedure. However, isolating and handling RNA ask for special precautions because naked RNA is highly susceptible to degradation by ribonucleases (RNases) (1). RNases are found everywhere and they are very stable and active enzymes that do not require metal ion co-factors to function and can maintain activity even after prolonged autoclaving or boiling. So that, all equipment and reagents should be treated to inactive RNases prior to use. Wearing gloves while handling reagents and RNA samples, changing gloves frequently, keeping tubes closed whenever possible and keeping isolated RNA on ice when aliquots are pipetted for downstream applications could prevent RNase contamination. We are used sterile, disposable plastic ware and they were RNase-free and do not require pretreatment to inactive RNAses (8-46,8-47). The quantity of the isolated RNA was determined by nanodrop spectrophotometry (absorbance at 260 nm of a 40ÂÂ µg/ml soluti on of RNA is 1.0) using nuclease-free water as a blank. A sample was reserved for quality assurance (see below) and the remainder was stored at -80 degree centigrade for one week. Gel electrophoresis (quality assurance of RNA) : The quality of the isolated RNA was verified by agarose gel electrophoresis. The gel was run at 100V for 30 minutes and photograph under UV transillumination. DNA digestion: A DNase digestion step was performed as a precaution using RQ1 RNase-free DNase kit although the TRIzol method generally results in RNA which is essentially free from genomic DNA (2). A sample was reserved for reverse transcription (see below) and the remainder was stored at -80 degree centigrade. Reverse transcription: 11 ÂÂ µL of DNase-treated sample was reverse transcripted, using Superscript II reverse transcriptase, to complementary (cDNA) by random hexamer priming.

I have chosen to analyse two Wordsworth poems, “Composed Upon Westminster Bridge” and “The World is Too Much With Us”

At one stage in his life, Wordsworth was greatly influenced by William Godwin, a philosopher who claimed that salvation lay only in reason perfected by education. Wordsworth adopted these Neo classic views for four years until he nearly suffered a nervous breakdown. Neo classicist writers believed that poetry had to be â€Å"fancier† than prose; they did not think of nature as a teacher as the Romantic writers did, they thought that reason was the prime source of inspiration and emotion was inferior to thought and they thought that poetry should be about people in high society; humble life was contemptuously ignored. After four years Wordsworth turned his back on Neo classicism and turned towards Romanticism. Romanticism was the idea that nature teaches the only important knowledge to man. The next philosopher to influence Wordsworth was Hartley, who taught that the mind was a â€Å"blank slate† until sensation introduced ideas into it, that sensation was the basis of all knowledge. Wordsworth, in his preface to the Lyrical Ballads insisted that poetry should be about the evocation of emotion and the inculcation of awareness through the artistic examination of immediate experience – poetry should be about how we emotionally respond to our experiences. For Wordsworth, the earth was not a dead thing, but full of life, full of the breath of the infinite Being. Composed upon Westminster Bridge shows Wordsworth apparently appreciating the beauty of a great city – though it is characteristic of his love for solitude that the poem is set in the early morning, when there is no noise or bustle. It has a calm, slow rhythm, which could represent the river, which adds to the relaxed atmosphere of the poem. The World is Too Much With Us embodies one of the main ideas of Romantic poetry – that in our daily life we have lost touch with the renewing powers of nature. The slow rhythm adds to the sombre, bitter mood of the poem. Both poems are written in the form of a Petrarchan Sonnet, which is divided into an octave (eight lines) and a sestet (six lines) by the rhyme scheme abbaabba cdcdcd. The essence of the Petrarchan sonnet is the unequal relationship between the octave and sestet. This structure is one of observation and conclusion, or statement and counter statement. In the octave of Composed upon Westminster Bridge, Wordsworth seems to be praising the city of London but the turn after the octave is a shift of feeling that develops the subject of the poem by surprise to its conclusion. In the sestet, Wordsworth praises the beauty of the morning that envelops the city. What seemed to be a celebration of London is actually a celebration of the beauty of the morning. The octave of The World is Too Much With Us plays with the idea that we have sold our souls to the material world and in doing so broken our bonds with nature. The sestet offers a solution to the problem, to go back to a less cultured, out of date society, the Pagans, and worship nature. Wordsworth was writing during the Industrial Revolution when society was becoming increasingly reliant upon mass production and there was a growth of towns and cities. These circumstances obviously affected Wordsworth – the majority of Romantic poetry is about nature whereas Composed upon Westminster Bridge is about the beauty of a city in the morning. Wordsworth used simple, everyday language that ordinary man could understand. The title â€Å"Composed upon Westminster Bridge† could be a pun; the word â€Å"composed† might also represent how the poet is feeling. Wordsworth suggests that people would have no sensibility if they could â€Å"pass by/ A sight so touching in its majesty. † The words â€Å"touching† and â€Å"majesty† suggests how powerfully this scene affects him. The words â€Å"now doth† seem to suggest that this is a transitory moment of beauty that will soon pass. The simile â€Å"This city now doth, like a garment, wear/ The beauty of the morning;† hints that the poem is celebrating the beauty of the morning before man wakes up and pollutes it. The words â€Å"bright and glittering† make this sight seem valuable; the â€Å"b† and â€Å"g† and the repetition of the â€Å"i† sounds makes it sound crisp. Wordsworth then compares the city to the country saying that â€Å"Never did sun more beautifully steep/ In his first splendour, valley, rock, or hill. † The awe-inspiring image of this moment touches him and he feels that it is the most beautiful thing he has ever seen. He uses the metaphor â€Å"that mighty heart is lying still,† as when the people have woken up the city is beating and pulsating. The overwhelming nature of the scene resonates within him and he teaches us that we too should be still and appreciate nature. What at first seemed to be a celebration of London is ostensibly a moral lesson on standing still and appreciating nature. Wordsworth was writing The World is Too Much With Us during the Agricultural Revolution when meadows and woodlands began to disappear. The title â€Å"The World is Too Much With Us† means that we are too concerned with the material world. Wordsworth uses the image of â€Å"getting and spending† to show the greed of our society. Our â€Å"powers† are our ability to appreciate nature and what she has to offer. â€Å"Little we see in Nature that is ours,† seems to suggest that if we don't own it, we don't want to know about it. â€Å"Sordid boon† suggests how bitter Wordsworth feels about how we have â€Å"given our hearts away† to the material world and betrayed nature. Wordsworth uses sensual imagery – â€Å"This Sea that bares her bosom to the moon,† and the simile describing the wind â€Å"like sleeping flowers† helps to add to the sombre mood of the poem and effectively describes nature as a thing of beauty. For our ignorance, we are â€Å"out of tune;† we are lost in the wrong environment and have lost the harmony between nature and ourselves, man is trying to dictate to nature. Wordsworth would â€Å"rather be a Pagan;† he would rather go back to an out of date society to become less cultured but closer to nature. He feels â€Å"forlorn† because he is out of tune with nature, he has to reject cultured things and be a Pagan to be truly happy. Proteus and Triton were sea gods in the â€Å"outworn† beliefs of Greek mythology. Wordsworth finds it appealing that the Greeks had gods of nature. This shows how they revered and appreciated nature. The poem teaches us that we should be getting back to nature instead of concerning ourselves with material possessions. Both poems address the same aspect of Wordsworth's poetry – that we have become desensitised towards nature and don't stop long enough in our daily lives to appreciate the beauty around us, instead we are concerned with â€Å"getting and spending,† although they do so in different ways. Like most of Wordsworth's poetry they both comment on man's complex relationship with nature and the world around us. Composed upon Westminster Bridge celebrates the beauty of the morning and how it can make the city of London more beautiful than â€Å"valley rock or hill† and how man cannot appreciate this whereas The World is Too Much With Us describes how we â€Å"lay waste our powers† and destroy nature.

Samples of Essay Format - What Is It?

Samples of Essay Format - What Is It? What you should do is to go over the topic. There are a few sorts of essay papers that have thoughts and purposes. When you compose an essay, bear in mind there are rules, guidelines and things you need to take care of during your essay writing. The lengthy essay is not simple to write since there are lots of things you will need to look at. You may use the exact standard format to compose your essay, regardless of the topic or whether you've got to provide your opinion or describe the benefits and disadvantages. You've got a guide and get ideas about what to do with your essay. Also, you can look for sample expository essay topics so you will be aware of what to research and that which you will deal with. The topics for argumentative essays are frequently very self-explanatory they're common understanding. A Secret Weapon for Samples of Essay Format Essay writing is a typical and extremely important thing in your academic life. Writers don't stick to a specific side. Writing essays is part of everybody's life and it's vital for the people to understand about how to compose an effective essay. Writing an essay for a student at an American University is something that's treated with terrific importance by a complete bunch of people which were also told to compose essays as students. Term papers constitute an extremely huge portion of the semester grade. It is not hard to write GMAT essay in case you have a guide and you've completed a good researched. If you understand what you will create GMAT essay, be sure you search the net and read more books. An essay, or a paper, has a huge set of rules that you have to follow if they'd love to have a fantastic grade. The Birth of Samples of Essay Format When you've done that, if you're prepared to go more in-depth, here's a step-by-step look at the way to format your organization proposal. The samples will provide you with ideas on the way you answer the questions offered to you. There's no greater approach to do so than to write. There are a couple of ideas you can utilize to make the procedure go well. Naturally, you may use lengthy essay samples, but you've got to understand main ru If you read the official guidelines about extended essay, it is important to take note of it since it'll completely help you. Examine the essay for plagiarism An outstanding essay is an exceptional essay, therefore a check for plagiarism is an incredibly important stage. Each of these kinds of paper is owned by the category of informative essay. Your extended essay introduction must be catchy. Essays aren't supposed to be easy for everyone. An essay is a fairly brief bit of writing on a specific topic. It will fit in the right amount of Letter sized paper. Structuring your essay in a correct format is essential. The essay will test the student wisdom and ability in answering the questions and analyzing the situation. The essay cannot be written according to standard essay scheme on account of the reason that the thesis statements in addition to conclusions are blurry. It's important to get a way of organizing your long essay. You have to compose a reflective essay that is simple to comprehend and you can do it by employing simple language. Samples of Essay Format Features Whenever you do write a paper, remember to don't do it in a format that is hard to follow by the reader that doesn't care about doing it. Make sure you essay is connected to politics culture or education. Make sure it can be read easily for the readers to understand what you are speaking about . Most readers will appear at an abstract first to see whether the paper has information they need to read. The response paper format is quite important to create your response paper accurate and to satisfy academic writing demands. After you've started, you will also have something down on paper or on screen you can improve on. Before you even begin writing your literature paper you've got to find out the principal point which will be discussed throughout the job. Thus, you commit to writing a paper of mediocre quality because you believe's the best that you can do with the time you've got at hand.

The Transformation Of A Hero - 1787 Words

Sankalp Nigam Mrs. Phillips English 9H Period 2 17 February 2015 The Transformation of a Hero â€Å"Life asks us to try things, make mistakes, and commit to a path of growth and maturity. Anyone can get old, but growth only comes with effort and commitment† said Phillip Humbert. Humbert tells people that the only way to grow and transform is by trying new things. One example of the path is the hero’s journey, which is a procedure of events that lead to a change in a person. This is what happens to the hobbit J.R.R. Tolkien establishes in his fantasy novel, The Hobbit. Bilbo Baggins is a well-to-do hobbit that is not keen on adventure and enjoys the comfort and safety of his home. Bilbo is just an ordinary hobbit living peacefully under the hill in his hobbit- hole until he is asked to join an adventure. He has two adverse personality traits from his ancestors and experiences dissonance between the contradictory characteristics. One of his ancestors are the Baggins, who are respected people and never do anything unexpected and don’t have adventures. On the other hand, he also possesses traits from his Took ancestors, who love adventure, but are less respected even though they are richer. One day Gandalf asks Bilbo to join him on a quest to retrieve jewels from a dragon’s cave. Bilbo immediately declines the offer, but begins to contradict his own thinking which leads him to eventually join the voyage to the Lonely Mountains. Bilbo’s Took side proved dominant over his Baggins sideShow MoreRelatedThe Transformation of the Hero in the Film, Raiders of the Lost Ark1108 Words   |  4 Pagesï » ¿Transformation of Hero in Raiders of Lost Ark Raiders of the Lost Ark is the 1981 film that follows renowned archeologist and professor Indiana Jones as he races against time and Nazis to uncover one of the greatest treasures believed to be in existence, the Ark of the Covenant. 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