Question

Question 5 (alternate). Gene and protein expression (Total 4 marks)

A. You have successfully completed your two-channel microarray experiment and have obtained Red and Green intensity values for each feature/probe on the array.

i. Describe how you would initially transform the data so that increases and decreases in gene expression are treated equally. (1 mark)

ii. What method would you use to normalise the intensities of the two dyes, as well as to eliminate other systematic differences due to unequal experimental conditions. (1 mark)

B. You wish to normalise data values between arrays. Provide details of a method for normalising data between arrays.

Answer 1

A) i)The hybridization data of the labeled cDNA is allowed to bind on the mRNA obtained from the cells of the normal samples and affected disease patient samples. The signals of the intensity of hybridization are recorded on the microarray screen. The data is normalized and the values are given a scaling of 1 for total strong hybridization and a value of 0 for a very low hybridization levels. The samples include a negative control for background intensity and a positive with intensity analysis of the binding for comparison with the diseased samples. The genes which show a high signal intensity of hybridization are collected for specific gene analysis in the diseased samples as compared to the normal samples during the experimentation studies. The histogram plots are made of the intensity of labeling of the genes with analysis of the p-values.

(ii) The intensity of the two dyes is captured on the scanner. The intensity of reading is then converted into a numerical reading with the help of a sophisticated software. The intensity is measured with the help of the shape, size and intensity of the microarray spot obtained after hybridization. The background noise signal intensity is subtracted. The ratio of the two dyes is calculated by analyzing the intensity of the signal obtained and then dividing it by the normal standard control intensity of the signal obtained in the microarray experiment.

B)The differential gene expression method is used for analyzing the data obtained in two microarray experiments. The intensity of red and green signals is calculated as a log value of R X G and compared with log value of R/G. The z-distribution and scatter plot are sketched. The intensity of samples labeled as red and green are again used and the samples are labeled as green and red accordingly. The standard deviation and mean are calculated and the values of standard deviations are analyzed whether they are one standard deviation away from mean, two or three standard deviation away from the mean. It is a filter method of analyzing the intensity of samples.