Relative and Absolute quantitative Real-Time PCR (qRT-PCR)
Introduction
Quantitative Real-Time in PCR (qRT-PCR)
The polymerase chain reaction (PCR) is a revolutionized technology used in molecular biology for detection and amplification of DNA generating thousands to millions of copies of a particular DNA sequence. Quantitative real-time reverse transcription-polymerase chain reaction PCR (RT-QPCR) is regarded as the golden standard technique in molecular biology and has been seen as a bench-marking analytic for DNA and mRNA detection, this technique is also used in a wide variety of bio-analytical science areas (Burns et al, 2005). RT-QPCR is a technique that simultaneously monitors, amplifies and quantifies nucleic acid accumulation in real time and is characterised as a high quality performance technique associated with high throughput, reproducibility, specificity and sensitivity. There are a number of steps involved in qRT-PCR which include RNA extraction, cDNA synthesis, PCR amplification and normalization with suitable internal standard or reference gene. Different selection methods, internal standards all have an effect on the reliability of qRT-PCR (Zhang et al, 2012). RT-qPCR validates and analysis data in basic PCR fluorescent thermal cycle software which depends on either absolute or relative quantification of PCR data, this validation system requires the comparison of sample to standard DNA or to reference genes, respectively (Roussel et al, 2007).
Relative quantification Real-Time PCR (qRT-PCR)
Relative quantification has been widely studied in cytokine or enzymes to evaluate gene expressions changes; this type of quantification omits the use of standards and allows the comparison of two experiments which...
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...e RT-PCR quantification of Kuruma shrimp transcripts: A comparison of relative and absolute quantification procedures'. Journal of Biotechnology, 129 (3), pp.391-399.
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Moreover, the class average curve shows a similar trend, as the curve flattens, at 70% but with an enzyme activity of 5.3 x10-3 seconds. This indicates that even though the saturation point is the same it was considerably lower than our results, which could indicate sources of systematic error in the design of the practical.
After 5 days of growth each slant was tested using the gram staining technique to confirm the complete isolation of the bacteria. Both isolations were completely successful. Then each sample of bacteria was subjected to a series of tests for identification.
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
When the PCR technique is completed, the tubes are stored at 4°C until analysis of the tubes. To analyze the PCR results with the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel for the electrophorese consists of 1.5% agarose gel with 0.5x TBE and 200ng/ml ethidium. bromide. The sand is a sand.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
The North American brine shrimp goes through several stages in development before reaching adulthood. The brine shrimp is first encased in a protective capsule within a female brine shrimp’s brood sac (Drewes, C, 2006). Here, egg development rapidly...
10. Sampaio-Silva, F., Magalhães, T., Carvalho, F., Dinis-Oliveira, R.J., & Silvestre, R. (2013). Profiling of RNA Degradation for Estimation of Post Morterm Interval . PLOS ONE, . Retrieved , from http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0056507 doi:10.1371/journal.pone.0056507
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Dinoflagellates are one of the four main types of phytoplankton, which are photosynthetic, single celled and free living organisms in the ocean. Dinoflagellates cause the Harmful Algal Blooms (HAB) also known as the red tide effect (Hackett et al 2004). Toxicity persisting at upper levels of the food chain is detected in them from the ones which are toxic, but not all such blooms are toxic. Enhanced detection capabilities may in part contribute to observed high frequency and severity of toxic blooms. As they are also important in the health of coral reefs their study has gained significant interest. Species are often selected for genome sequencing based on their importance as a model organism or relevance to human health, such as the HAB case.
In the first study examined, “Effect of Different Salinities on the Survival and Growth of Artemina Spp,” researchers Soundaraparian and Saravanakumar designed an experiment to ascertain the ideal conditions for the growth of brine shrimp, or Artemina. In the Introduction, the scientists note the growing significance of Artemina, as it is now used as live feed for over 85 percent of cultured species around the world. Thus, a demand to grow huge quantities of Artemia has arisen, making this study incredibly relevant.
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The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...
Sharing certain aspects of practice with other disciplines of pathology like clinical pathology, anatomic pathology, biochemistry, and molecular biology, molecular pathology seeks to understand and diagnose, at a molecular level, the mechanisms and origins of diseases (Harris and McCormick 2010). Through patient samples tests are carried out to measure