In a previous article we explained the principle upon which the Microarray technology and they are coloring the mRNA or cDNA for each type of cell in red or green and then compare the ratio of the difference sea shell shop between the percentage of radiation gene in both cases, we choose the genes that showed a significant difference between the samples. We have seen that there are two types of these chips, chips manufactured using Android (Spotted Array) chips are manufactured using optical and X-ray masks using method of In-sito spotting, and represented the habit in Affymetrix chips, called this type of chips Pal oligonucleotides ships.
Unless transported sea shell shop in a previous article is that in contrast to the chips manufactured using Android, the use of one type of cells in each experiment in the Affymetrix chip, which means that it must do Btgrepettan (one using normal cells and other cells using the patient). This launches label chip dual channel (Two channels array) sea shell shop on chips that allow the use of two colors at the same time and label single-chip channel (Single channel array) chips that allow the use of a single color.
In this article we will focus on ways to analyze this data and what are the precautions that must be taking into account at every stage. Usually do stages are divided Microarray experiments sea shell shop to seven key stages as shown in the figure.
1. Biological question: even if this stage intuitive, but in my opinion is one of the most important stages if the researcher must experience before embarking on knowledge of the type of problem that wants to be answered, for example: What type of disease? What are the characteristics of the samples must be taken? sea shell shop What is the situation in which samples must be in it? .... Etc.. Identify the target at this stage contribute to an increased sea shell shop probability sea shell shop of obtaining the desired sea shell shop results and understanding of the results of the experiment sea shell shop and correction of errors, if any. 2. Trial design: It is during this phase thinking about the details sea shell shop of the experiment and sample preparation. With low price chips now advisable sea shell shop to use several sea shell shop samples of types (for example, five samples of healthy cells and five samples sick) in order to increase statistical precision and the abolition of errors in each sample, where it is well known that if we re-experience using the same sample and the same amount and use the same device The same person will not experience the results will be matched 100%. For example, take a sample from one patient a large amount somewhat we Baksimha sea shell shop into equal amounts and we experience every time you use one of the copies. You can also put several copies of the cDNA of the gene in the same slide. Technical repeats allow us to know the error rate at the level of one type of samples. For example, three samples are taken to normal cells from three healthy volunteers. From the statistical sea shell shop side allows us Almtkart the biological knowledge of the error rate at the level of the community samples. In addition to this you must also specify the type of chips that will be used, you must use chips dual channel or single-channel chips. In the case of the use of thin bilateral Alguenat should only take into consideration that the rate of hybridization and the mRNA ratio will be different between the two samples, even if the percentage equal to the percentage sea shell shop of radiation color red (Cy3) and green (Cy5) are not equal. To correct this problem, we are the heart of the process colors, where we experience twice in each experiment turn colors between Alaintn, فالعينة which was painted red in automated discoloration experiment in green and sample colored green in the first experiment in red discoloration and returned the experiment.
4. Image processing and calculating the proportion of activity: at this stage, using specialized sea shell shop programs in order to determine the percentage of radiation in both probe and determine the percentage of jamming caused by radiation from sources other than the hybridization process. As there are several factors that can affect the rate Radiation kind كوضعية, room pressure, ... Etc.. For this, you must first delete these disturbances background (Background Signal) and maintaining reference real as possible. Different programs used in image analysis, but all work according to the principle of one is for each probe point is determined by the most intense and calculate the proportion of radiation. After ذالك be deleted noise ratio. Let $ R $ initial ratio of radiation red Let $ G $ ratio of the initial green radiation. After calculating sea shell shop the proportion of disturbances sea shell shop background $ R_b $ for Red and $ G_b $ for green is calculating the proportion of activity (expression) gene deletion ratio jamming becomes proportion of radiation equal to $ R-R_b $ for Red and $ G - G_b $ for green and we can calculate the forgotten change $ M $ are as follows:
Image shows the principle of the work of some image processing software (source) for the single-chip design different channel where it is kind design Msparin each gene. Probe, the first contains a series matching a series of gene (Perfect Match) and symbolized her with $ PM $ and a series of different (Missmatch) and symbolized her b $ MM $, the principle here is that the radiation resulting from the process of hybridization in the probes of the series matching the proportion of real activity and radiation output on the contrary series represents the confusion sea shell shop and the goal is to be the difference between sea shell shop these large Alahaain, equal If it means that the measurement is incorrect. Usually measured by the proportion of difference between Alahaain the average change between each pair of probes Bstamal the following sea shell shop statement
5. Mqais quality: After calculating the proportion of each gene radiation and calculate the value of jamming back it is advisable to do measuring the ratio of the quality of the data to know you can continue to use them and see which
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