Multiplexing Of Singleplex Real Time Pcr Assays Into Duplexes And Triplexes

Inhaltsangabe:Introduction: Breast Cancer: Cancer describes a group of various diseases, where cells start changing their molecular structure and begin to grow and to supersede normal cells. Cancer is induced by numerous different elicitors, which finally all lead to an interference of the genetically regulated balance between cell cycle and apoptosis. Although every organ in the human body can be afflicted with cancer, there are significant differences in frequency relating amongst others to age, sex, geographic region and personal habits. In the industrialized countries, breast cancer is the leading cause of death for women at the age between 30 and 60 years. With estimated 636.000 incident cases in the developed countries and 514.000 in the developing countries, breast cancer is the most prevalent cancer type among woman worldwide. Once detected, the cancer is classified based upon pathological characterizations of the tumor or a biopsy and the lymph nodes. A clinical way of characterizing the tumor is the TNM-classification, which describes the size of the tumor (T), the number of affected lymph nodes (N) and the existence of distant metastases (M). The histological classification characterizes the carcinoma according to its structural and cellular appearance and the amitosis rate leading to a grading from 1 to 3. An immuno-histological examination provides information about the estrogen- and the progesterone-receptor- and about the Her-2/neu-status. Breast cancer is a very heterogeneous disease. There are basic classifications that are unquestioned, even today. Recent studies confirmed the need to determine well known markers (i.e. estrogen (ER) and progesterone (PR) receptor status or HER2 status), but the large variety of subtypes and the corresponding different molecular pattern impede a uniform treatment. Although already today other factors than anatomical classifications are being taken into consideration, there exists a need for further biological markers to assist the physician in charge with his evaluation. Beside the diagnostic recognition, the choice of appropriate therapy and the prediction of prognosis are goals that should be reached in order to prevent early stage cancer patients from therapies that provide minimal benefit but reduce their quality of life by intense adverse reactions. RNA Expression Profiling and Prediction: Already today there are numerous genes associated with breast cancer occurrence, therapy [...]

Das Werk beschreibt die Optimierung eines prognostischen molekularbiologischen Tests für die Brustkrebs-Diagnose und -Therapieentscheidung. Im Rahmen der Arbeit wurde die Zahl der Reaktionsräume für Polymerasekettenreaktionen (PCR) durch die Optimierung vom Multiplexen derart reduziert, dass parallele und kosteneffizientere Analysen möglich wurden.

An increasing number of genetically modified organisms (GMOs) continues to be produced every day. In response to the concerns raised by the development of GMOs and their incorporation in foods and feed, guidelines and regulations to govern and control the use of GMOs and their products have been enacted. These regulations necessitated the design of methods to detect and analyse the presence of GMOs or their products in agriculture produce, food and feed production chains. Design of techniques and instruments that would detect, identify, and quantify GM ingredients in food and feed will help inspection authorities to relay reliable information to consumers who might be concerned about the presence of GM ingredients. Information generated by detection of GMOs in food and feed would be helpful for setting regulations that govern the use of GM components as well as for labeling purposes. Qualitative detection methods of GM-DNA sequences in foods and feeds have evolved fast during the past few years. There is continuous need for the development of more advanced multi-detection systems and for periodic updates of the databases related to these systems. Testing and Analysis of GMO-containing Foods and Feed presents updates and comprehensive views on the various methods and techniques in use today for the detection, identification and quantification of GMOs in foods and feed. The eleven book chapters cover recent developments on sample preparation techniques, immunoassays methods and the PCR technique used in GMO analysis, the use of biosensors in relation to GMO analysis, the application of nucleic acid microarrays for the detection of GMOs, validation and standardization methods for GMO testing, in addition to the type of reference material and reference methods used in GMO testing and analysis. Some of the ISO standards designed for identifying and detecting the presence of GM material in foods are also presented in the book.

This laboratory manual includes the latest tools and techniques involved in genomic research. It starts with an introductory chapter on genomics and the various tools and applications involved. The initial chapters present protocols for basic techniques such as DNA isolation, electrophoresis, PCR, cDNA synthesis etc. The book then goes on to describe more advanced techniques such as next-generation sequencing, exome sequencing, use of RNAi, RNAseq, genome editing, single cell genomics etc. Each topic includes a brief description, information on the principles involved, materials & methods, protocol, and expected results, with diagrams and graphs. All protocols are presented in a very lucid and precise way, to make it easy for readers to follow and replicate them.

Future pandemic prevention from influenza virus type A strain (H1N1) 2009 in India require rapid and reliable testing of larger samples collected in year round at different geographical, environmental and social strata for novel strains and its differentiation from seasonal ones. In this study, a real-time Reverse Transcriptase PCR (RT-PCR) assay was used in multiplex for detecting Influenza A gene, Swine influenza H1 gene and human RNP gene, with normal PCR in singleplex for B viruses for 24 patient nasal swab samples, who were reporting with influenza like illness (ILI) from local clinic in Chennai and these patients showed low circulation of pandemic (H1N1) 2009 strain. KEYWORDS: Influenza, Nasal Swab, Real-Time PCR, RT-PCR, Swine Flu, (H1N1) 2009 Strain

Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur and Doty (1961) first showed that denaturation of the double helix could be reversed - that the process of molecular reassociation was exquisitely sequence dependent. Gillespie and Spiegelman (1965) developed a way of using the method to titrate the number of copies of a probe within a target sequence in which the target sequence was fixed to a membrane support prior to hybridization with the probe - typically a RNA. Thus, this was a precursor to many of the methods still in use, and indeed under development, today. Early examples of the application of these methods included the measurement of the copy numbers in gene families such as the ribosomal genes and the immunoglo bulin family. Amplification of genes in tumors and in response to drug treatment was discovered by this method. In the same period, methods were invented for estimating gene num bers based on the kinetics of the reassociation process - the so-called Cot analysis. This method, which exploits the dependence of the rate of reassociation on the concentration of the two strands, revealed the presence of repeated sequences in the DNA of higher eukaryotes (Britten and Kohne, 1968). An adaptation to RNA, Rot analysis (Melli and Bishop, 1969), was used to measure the abundance of RNAs in a mixed population.