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In order to study the advanced prostate cancer, mouse was used where the cells from the tumor were injected into the mouse. The basis idea was to study the metastasis of prostate cancer. The process took great amount of time, as cells were not developed. However, with the growth of cells, there was success in terms of metastatic tumor cells being obtained.
The first question is based on characterizing the biochemical changes largely associated with the acquisition of the metastatic phenotype. The question aims at seeking answer for describing two different strategies that would be helpful in identifying genes with altered expression in metastatic cells compared to the cells of the primary tumor. In this regard, the DNA microarray analysis and Reverse Transcription Polymerase Chain Reaction (RT-PCR) have been used as the ideal strategies.
DNA Microarray Analysis
The best thing about DNA microarray is in the form of being easily available requiring no effort to develop one. In addition, these available chips have a number of sports and each spot has unique and multiple copies of similar DNAs and are always single stranded. It needs to be mentioned that the molecular and cellular mechanisms that are involved in the progression of prostate cancer towards the metastatic phenotype are not well defined and understood. However, when these cells with metastatic potential are analysed using the microarray analysis offers great deal of insight on identifying genes that are related to the metastatic phenotype. In this regard, affymetrix arrays were used to understand the gene expression of rat’s cell lines defining the prostate cancer. At the same time, MAT-LyLu and G microarray data were used to understand and analyse the pathway and functional group. Furthermore, selected genes were subjected to polymerase chain reaction for validating the results being obtained from the microarray data in a significant manner. Findings were quite interesting in nature suggesting the fact that there were different expressions of genes from a number of signaling and wide arrays of metabolic pathways.
At the same time, over expression was observed in many genes with equal number of under expressions. Genes were further categorised into the cell motility, transporters, matrix interaction, and cell proliferation and many of them were previously not associated with the phenomenon of prostate cancer. This also helped in understanding that genes with altered expression being related to metastatic prostate cancer can be easily identified but further validity in regards to human prostate samples will define the usefulness as biomarkers in early detection of metastasis prostate cancer.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
This strategy is based on understanding the production of cDNA from mRNA that is followed by increasing of the cDNA through the chain reaction. The mRNA was extracted from the tumor cell and also metastatic cell being kept in two separate tubes. The next step was addition of PCR to RNA and the samples were incubated at high temperature of 390 C for the synthesis of DNA for a period of one hour. The mixture was kept at different temperature like 950 C for denaturation, 650 C for annealing and 750 C for amplification. This process was repeated for more than 40 times until the sample was aided by the agarose gel electrophoresis. This helped in generating digital images that were analysed using the software showing the fact that cDNA produced from metastatic sample were high in amount compared to the primary tumor sample.
Advantage and Disadvantage
It can be believed that the major advantage of DNA microarray is in the form of comparing numerous genes in a simple and precise manner whereas the RT-PCR is assumed as the best test of qualitative gene expression and also helping in the quantification of the cDNA. Disadvantages of DNA microarrays is in the form of shorter shelf life while that of RT-PCR is in the form of too much of time required to validate the findings as it is majorly based on the chain reaction completion period.
It needs to be mentioned that different genes contribute to metastasis in a different manner and the same is applicable in regards to contribution indifferent group of tumors. Elevated biomarkers might suggest metastasis in the form of IL-6 elevation and fusion gene can be assessed based on the analysis of genome translocation. For tumor suppressor gene, the analysis of p53 is important in order to understand whether it is suppressed or not. For gene silencing, DNA methylation can be assessed and analysed. There are a number of in vivo and in vitro assays that can assess the role of gene product in metastasis.
This means cells can be collected from the primary tumor and metastatic site along with the opportunity of growing them separately to assess the growth. There are large degrees of evidences supporting the fact that metastatic cells grow faster that the tumor cells. It is widely found that metastatic cells are known for invading the media and penetrating the porous membrane to get inside the serum at a high rate and speed that primary tumor cells fail to do. Thus, cultures cells can be kept in a media being separated from the serum by the porous membrane to test the growth of different cells in a systematic manner. For in vivo studies, it is important to have practical understanding of the phenomenon and mouse can be used as a model for prostate cancer to observe the metastasis. Genetically modified genes need to be inserted for identifying the tumor growth in order to ascertain the nature of metastatic cells that are known to be highly proliferative cells.
Many findings have also suggested that metastatic cells are highly infiltrated compared to the tumor cells and can be identified using in vivo and in vitro phenomenon. It needs to be mentioned that in vivo studies in regards to human beings can be further conducted based on the results ascertained from the tests conducted on mouse. It can be further said that both in vivo and in vitro studies are important to important to understand whether these genes make a general contribution to metastasis in a wider group of tumors. This also means that there are a certain advantages and disadvantages associated with the same that needs to be discussed.
Advantages and Disadvantages
It can be further said that in vivo assays are helpful in understanding the role of gene products in metastasis and often can be responsible for identifying the gene responsible for genetic alterations. The results can be further used for the treatment of prostate cancer in human beings based on the successful findings. On the other hand, in vitro studies are helpful in ascertaining useful findings in the scientific settings that will be helpful in understanding the over and under expression of a certain gene. However, validity in both the settings is the major difference as in vivo findings might result in better findings. However, in vivo findings are expensive in nature requiring great amount of time.
It needs to be mentioned that the two distinct experimental strategies for identifying the proteins that might interact with the protein are in the form of Yeast Two Hybrid Assay and Tandem Affinity Purification- Mass Spectrometry (TAP-MS). The two have been discussed in terms of the best experimental strategies for identifying proteins.
Yeast Two Hybrid Assay
There is no denying that there has been significant improvements in Yeast two hybrid system since last two decades that has revolutionized the way protein interactions are identified and detected. This experimental strategy is based on the reconstitution of the functional transcription factor when two proteins interact with each other. This interaction usually happens in genetically modified yeast strains where the transcription of a reporter gene leads to the generation of a specific phenotype. This growth happens on a specific medium being defined by the change in the color of the yeast colonies. The most identified and popular reporter genes are in the form of HIS3 to select yeast on a medium histidine and LacZ that is used to screen yeast in a colorimetric assay.
The technical details in this regard are quite important where two fusions in the form of hybrids are constructed between each protein or DNA Binding Domain (DBD), or Activation Domain (AD) of the transcription factor. In this regard, the protein infused to DBD is termed as “bait” whilst the protein infused to AD is termed as “prey”. The interaction between the bait and prey enhanced the proximity level of DBD and AD that leads to the reconstitution of a functional transcription factor. There are different kinds of DBD in the form of LexA and Gal4 that can be used. It needs to be mentioned that as a genetic technique, this method offers a sensible and cost effective approach to test and analyse the interaction between the targeted proteins in a significant manner. It is also useful in using favorite protein as bait for screening the libraries of proteins prepared from different and desired cell types or tissues.
Tandem Affinity Purification- Mass Spectrometry (TAP-MS)
TAP-MS is a method that is used for rapid purification of protein complex under different conditions. The method involves fusion of the TAP to protein followed by two-step affinity purification process. This method further relies on information of the interested protein and specific cell line only in order to create fusion protein embedded with purification to reduce the amount of binding. This method is also useful in combination with mass spectrometry in large-scale approach for systematically analysing the proteomics. There is no doubt that majority of the biological processes are driven by multi protein complexes rather than any specific protein and identifying the protein complex is quite important for gaining an understanding on cells and organisms. This method is quite useful in fast and effective purification of proteins along with minimising the background. This also helps in identifying even low abundant protein complexes using mass spectrometers. Overall, this method has revolutionized the way of dealing with multi proteins along with helping in gaining molecular understanding of different cells and organisms in a systematic manner.
Advantages and Disadvantages
The major advantage of TAP-MS is in the form of real time determination of proteins in vivo without prior knowledge of the composition. It is also simple and widely acceptable along with offering high yield. On the other hand, the Yeast Two Hybrid System is an easy and cost effective approach that has been practiced from decades. Disadvantages of TAP-MS are in the form of analysis of data that is quite challenging along with contamination of proteins when they are abundantly present. On the other hand, disadvantages of Yeast Two Hybrid System are in the form of less numbers of posttranslational modifications and other binding factors.
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Uniresearchers is a leading team of researchers in the field of academic writing. With the track record of delivering 500+ high quality dissertations and 2500+ essays, assignments and coursework’s Uniresearchers has always tried to keep up with the expectations of our clients.
