You have recently isolated and cultured a cancerous cell line from a brain tumor of a patient. You are interested in determining what may be different at the gene expression level between the cancerous cellular tissue and the normal tissue. Design an experiment to characterize the differential gene expression between the two cell types.
For this purpose you can use at least three different approaches: Microarray, RT-PCR or RNA-seq. Firstly, you need to specify expression of what genes you need to know. Secondly, you need to estimate whether you need absolute quantity of the desired mRNA of your selected genes or relative. Therefore you will choose a way for statistical analysis of your data in advance. In RNA-seq the expression signal of a transcript is limited by the sequencing depth and is dependent on the expression levels of other transcripts, whereas in array-based methods probe intensities are independent of each other. Using RNA-seq or microarray you will have a massive expression data of many different genes. Later, using bioinformatics it is possible to estimate relative aboundance of the investigated mRNA related to the control. For perfoming real-time PCR you need remember to isolate RNA, to design the primers for the genes of your interest. For all cases, you need to specify expression of what genes you need to know. That is how to identify the differentially expressed genes in two or more conditions in the cancerous cellular tissue and the normal tissue.