Increasing Student Achievement through Preventing School...

Increasing Student Achievement through Preventing School Violence Introduction School violence is any type of violence, whether it is a simple threat on another student or a school shooting, that happens in the school environment. Student achievement is the efforts made by the student to work hard in the classroom. The purpose of this research paper is to examine how school violence impacts student achievement. It would seem that school violence would have a negative impact on student achievement. If this is the case then we need to do what we can to prevent violence in schools. Technology can be used to help control school violence. Information on School Violence Violence in our schools has become an increasing†¦show more content†¦It was found that males tended to fall victim to crime more so than females (Furlong Chung, 1995). Multi-victim students of violence were more likely to receive D’s and F’s whereas non-victim students tended to receive more A’s. Students have not experienced crime perceived their schools to be a safe environment and therefore were able to be more focused to their schoolwork than worrying about violence. Since they are able to focus better in school, their achievement level is considerably higher than a student who has experienced many incidents of violence. Multi-victim students of violence tend to be more worried at school and do not feel safe in their school environment. These insecure feelings leave the students paranoid and therefore are not able to focus on their schoolwork as well as non-victim student. In order for a student to be successful we need to eliminate all distractions from the school environment. Violence is a distraction and even though it can not be eliminated completely, we need to see to it that it we remove as much of it as we can from our schools. All students deserve the right to a safe school and to have an equal chance to be successful in life. 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Are You Really My Hero - 1216 Words

AJ Wells Professor Carly Smith ENGL 112 80N-N1-201420 13 December 2014 Are You Really My Hero? â€Å"Heroes have their scars. Some you can see, some you read about later on.† George Foreman Growing up we all had our dreams of winning the gold medal in an Olympic event, scoring that game winning basket, being a prize fighter, or hitting the homerun with bases loaded in a World Series. We imagined the feeling of success and glory but we also realized that few do accomplish such. Seeing such deeds are what creates our desires, determination and goals. The athletes that achieve such give us our heroes to aspire to attain such levels. We want the future generation of athletes to have these same dreams, goals and hopes of success that we grew up with. But are our current athletes the type of heroes we want our children and young athletes to look up to? Performance Enhancing Drugs are becoming the norm for many of our athletic heroes and modern day headlines are expounding on the wide spread usage among our elite athletes. Some authors such as Michael Lavin in â€Å"Sports and Drugs: Are the Current Bans Justified?† states that the expectations and demands place d upon today’s athletes are much greater than in the past and this pressure leads to using enhancements. Professional athletes are now paid large salaries so that more expectations are made of their abilities than may be achievable with natural workouts and practice of the past. There is pressure to stay in the gameShow MoreRelatedCharacteristics Of A Hero891 Words   |  4 Pages Tree traits of a hero â€Å"A hero is an ordinary individual who finds the strength to persevere and endure in spite of overwhelming obstacles.† - Christopher Reeve. Having heros in this world is really important. If we didnt have everyday heros our world would be a lot different. 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Counseling Ethics Samples for Students †MyAssignmenthelp.com

Question: Discuss about the Counseling Ethics Case of Susan Lim. Answer: Susan Lim is a prominent surgeon in Singapore who has performed several complex surgeries throughout her career. Due to her exceptional capabilities, she is famous all across the globe. She was head surgeon in the first liver transplant in Singapore, became the youngest person and first Singaporean to become the fellow of Trinity College, Melbourne. In 2001, she undertook the case of Pengiran Anak Hajah Damit, the sister of Brunei queen. In 2007, the patient died a d Dr. Susan Lim was accused of overcharging the patient with a very high charge. According to the media reports, Dr. Susan Lim was accused of making a bill of 40 million dollars during the period of 2001 to August 2007 (Asia One Health, 2016). In the later investigations, it was found that the bills constructed were astonishingly high than the normal circumstances. In 2013, doctor was prosecuted and found guilty. Singapore National Council filed a case against the Susan Lim and won the case eventually. The doctor was found guilty. During the judgment, the doctor presented her side by stating that she charged the patients in accordance to the services provided to them. She stated that although the prices were very high, but they were in accordance to the services. In order to excellent services to her client, she received training from all across the globe. In several instances, she herself paid for the services provided to her client such as sending the patient from Singapore to Brunei; therefore, she was only asking them from the client (John Harding, 2011). Moreover, her professional conduct can be scrutinized from the fact that in order to halt the legal processioning, she threatened the Singapore Medical Council that she can bring into light several unnecessary and embarrassing facts which can bring shame to the government and the royal family of Singapore. She stated that she knows the inconsistent relationship between Ministry of Health, Government of Brunei and the royal family of Brunei. The threat brought her a second disciplinary action. Identification of Ethical Dilemma The code of medical ethics regulation state that the doctors should not overcharge their patients. According to the professional ethics, doctors should charge their patient according to the services provided by them. However, same is not the case of Doctor Susan Lim. Moreover, overcharging is against the professional practices, as it refers to deceit and abuse of ones position. It can be critiqued that there was another moral issue wherein that the doctor tried to threaten the government to drop the accusations. Major Stakeholders of the Case: Upon the review of the ethical issue, the major stakeholders can be identifies as Brunei Royal Family, Singapore Medical Council, Singapore government and the doctor herself. Susan Lim Susan Lim was the primary stakeholder in the ethical issue. She was the performing doctor who took case of the patient for six years. She was of the viewpoint that she provided exceptional services to the patients, invited the foreign professionals and received training which made it acceptable for her to overly charge her patients (Youtube, 2011). Singapore Medical Council (SMC) Singapore Medical Council was also a major stakeholder in the case, as it is the responsibility of the organization to maintain the professional integrity among the healthcare professionals and doctors in the Singapore. Any breach of duty is against is breach of guidelines of the Singapore Medical Council. In the present situation, Dr. Susan Lim breached the guidelines of Singapore Medical Council and the organization should try to impose justified penalty for the unethical behavior (Broad, 2000). Brunei Family Brunei family is the victim in the present situation. The doctor exploited the family due to their excessive wealth and influence. The doctor tried to obtain as much fees as she possibly could as she knew that her client was capable of paying higher fees. When she faced trial, Susan Lim also tried to exploit the vulnerable relations between Brunei government and Royal family as she was aware of their relations (Timmons, 2012). Singapore Government It is the responsibility of the government to protect the society from any type of criminal or civil offence. The professional medical society of Singapore has made code of conduct for the healthcare professionals so that they work ethically. In the present case, the Singapore government is a stakeholder as it is its responsibility to maintain law and order in the country. Ethical Dilemma The ethical dilemma can be defined as a situation in which a person is conflicted between different courses of actions in which taking one action will result in transgressing some other moral imperative. In the present situation, the ethical dilemma of overcharging the patient has been identified. The doctor calculated the fees according to her perspective and presumed that the exceptional services provided to the client justify the fees imposed upon them. She singularly treated one patient and kept all her focus to the sister of the royal queen of Brunei (Garber, (2008). Therefore, in her perspective, the fees imposed upon the client were justified. However, the royal family of Brunei was displeased with the doctors fees as they were exceptionally high. They presumed that the doctor was charging them with high fees die to their wealth. Moral Theories In the recent years, a large number of moral theories have emerged to examine whether certain actions are morally fit or not. These theories try to provide a systematic method to evaluate whether certain actions are morally right or wrong. However, different moral theories are dependent upon different perspectives and several times contradict each other. In the present section, the ethical dilemma of Doctor Susan Lim is examined under different ethical frameworks. Utilitarianism Utilitarianism is the most popular and common ethical theory which states that the all those actions are ethical which have better outcomes for the society. The utilitarianism is a version of consequentialism and states that only the result of certain actions can be categorized as right or wrong. In this framework, the interest of all the stakeholders is considered equally. There are certain benefits and disadvantages of utilitarianism (Dreier, 2009). The advantages of utilitarianism are that it promotes equality and happiness of all the people in the world and assist the people in taking tough decisions. In most of the complex and tough decisions, the emotions and the desire of the people become predominant; however, adopting utilitarianism approach assist the people in thinking rationally and taking the right decision (Jacobs, 2008). When this theory is applied to the case of Doctor Susan Kim, it can be evaluated that the action is morally justified. The overcharging of fees was ethically wrong as it was only beneficial for the doctor. The client was wronged as the fees were astonishingly high and determined according to his affluence. Moreover, the action will be a bad influence on the society as it is a breach of the moral code of conduct of the healthcare professionals. Teleology The teleology theory of morality states that the morality of the actions depends on the expected outcomes of the actions. Teleology theory states that if a person is following a certain course of action to achieve good from the situation, then that action is justified (Hinman, 2012). The teleology theory has certain disadvantages like it allows immoral actions on the pretext of better consequences. The consequences of most of the actions are quite uncertain; however, it allows the people to take morally unjustified actions (Alexandra Miller, 2009). According to this theory, the actions of Susan Lim are morally wrong as she was not focused on the treatment of the patient rather than increasing the probability of charging her client in the maximum possible manner. Although she provided exceptional services to the patients, pricing her client highly was always in her mind. Counseling Process Doctor Susan Lim should be provided counseling on the moral justifications of her action. Adopting a morality framework and examining the actions through it can assist the doctor in taking morally right actions. Decision-Making Process In the daily life of professionals, it is important that the people should take morally correct and ethical decisions. In order to take morally correct and right decisions, the professionals need a decision making framework. In the present section, an eight-step decision making model is proposed which can assist doctor in ethical decisions in the near future. According to this model, the decision making process has eight phases. Phase 1: In this phase, the problem is defined. In good decision-making, the problem definition is very important. It assist person in establishing clarity on the actual problem and examining various viewpoint of different stakeholders. In this phase, the root or the underlying causes of certain problems is identified. In addition to it, in this phase the limiting assumptions, system and the interface boundaries and the stakeholder issues are identified (McKee, Kemp Spenxce, 2012). Phase 2: In the second phase of the model, the requirements for the acceptable solution are identified. The Acceptable solution must meet certain requirements and address the underlying problem. The requirements define what the solution must be capable of doing. Phase 3: In the third phase, goals and objectives are defined which can be achieved by the solution. It refers to the broad statements of intent and value with the decision. Phase 4: In the fourth phase, the person should ponder on different alternatives which can be used to address the problem statement. In good decision making, it is important to evaluate different alternatives according to their effective outcomes. In most of the cases, the alternatives are evaluated according to their ability to meet the requirements and goals of the organization. Phase 5: In the fifth phase, different criterions are defined according to which the alternatives to the solution are evaluated. It is important to define discriminating criterion which determines how well each goal is able to achieve the result. Phase 6: In the sixth phase, a decision making tool must be developed which is able to select the best route for the solution. The decision-making tool should be based on the complexity of the problem (Mowen, Hansen Heitger, 2016). Phase 7: In this phase, the selected alternative is implemented to achieve the solution. The person should also monitor the solution for its results. Phase 8: In the last phase, the implemented solution is measured for its performance. After the evaluation, the person can make amendments in the decision so that the desired outcomes can be achieved (Mowen, Hansen Heitger, 2016). Conclusion It can be concluded that the course of action taken by Doctor Susan Lim is unethical and immoral. It is due to the fact the doctor overcharged her patient excessively. The moral dilemma of Susan Lim has been evaluated according in utilitarianism and the teleological moral framework. According to the utilitiarism theory, all the actions are justified which have better outcomes for the society. In addition to it, the teleological moral theory states that if the intentions of the people are good then the actions are justified. The action can be categorized as immoral according to both theories. Upon the evaluation of situation, it has been determined that the action was immoral. The doctor can be provided counseling with the help of decision making framework. The eight step decision making framework has been suggested will be beneficial in the future decision making process of the organization. References Asia One Health. 2016. Surgeon billed Brunei patient $40m over 4 years. Retrieved December 10, 2016 https://health.asiaone.com/health/health-news/surgeon-billed-brunei-patient-40m-over-4-years Broad, C.D. (2000). Five Types of Ethical Theory. Psychology Press.Chew, R. (2011). DOCTORS FEES AFTER SUSAN LIMS CASE Implications for the Medical Profession. Retrieved December 10, 2016 https://www.sma.org.sg/UploadedImg/files/Publications%20-%20SMA%20News/4511/Insight.pdf John Harding. (2011). Dr Susan Lims threatening letter to Foreign Minister George Yeo. Retrieved December 10, 2016 https://johnharding.com/2011/03/dr-susan-lims-threatening-letter-to-foreign-minister-george-yeo/ Youtube. (2011). Singapore Top surgeon 'threatened' MFA - 28Mar2011. Retrieved December 10, 2016 https://www.youtube.com/watch?v=xWuWLuSZ95Y Garber, P.R. (2008). The Ethical Dilemma. Human Resource Development. Timmons, M. (2012). Moral Theory: An Introduction. Rowman Littlefield Publishers. Jacobs, J. (2008). Dimensions of Moral Theory: An Introduction to Metaethics and Moral Psychology. John Wiley Sons. Alexandra, A., Miller, S. (2009). Ethics in Practice: Moral Theory and the Professions. UNSW Press. McKee, A., Kemp, T., Spenxce, G. (2012). Management: A Focus on Leaders. Pearson Higher Education AU. Mowen, M.M., Hansen, D.R., Heitger, D.L. (2016). Managerial Accounting: The Cornerstone of Business Decision-Making. Cengage Learning. Dreier, J. (2009). Contemporary Debates in Moral Theory. John Wiley Sons. Hinman, L.M. (2012). Ethics: A Pluralistic Approach to Moral Theory. Cengage Learning.

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