In a RNA-seq study of tumor tissue from glioblastoma primary tumor tissue, a gro

Question

In a RNA-seq study of tumor tissue from glioblastoma primary tumor tissue, a group of up and down-regulated genes were identified (that is, the transcript levels were changed relative to control, normal tissue). Some of these genes represented known cancer genes, others were novel genes. Describe three (3) analyses you would conduct for only ONE of the up-regulated OR down-regulated (not both) novel genes. (That is, how would you determine the possible role of this gene in glioblastoma oncogenesis (if any). Remember, you would have the gene cDNA cloned and available vectors, glioblastoma cell lines, etc.) Include the following:

1. The three different assays to analyze your gene of interest. OUTLINE the steps for each assay. That is, how will the experiment be conducted? What type of cells will you use? What will be measured? What will be graphed? What are the controls?

2. The expected results for each of the assays.

Explanation / Answer

(i) If you cDNA of the glioblastoma cells you can do realtime quantitative PCR by using SYBERGREEN reagent to look for the expression of your gene of interest by using specific primers. For example If your RNA sec data is showing downregulation of any particular gene, in RTPCR You will get the low expression of you desired gene as compared to control cDNA at the mRNA level.

(ii) You can prepare the cell lysate of normal cells and glioblastoma cells and load the same amount of lysate in to a SDS-PAGE gel and do the transfer of this gel to the nitrocellulose membrane and probe it with the antibody against your gene of interest For example If your RNA sec data is showing overexpression of any particular gene, in western blot You will get the high expression ( prominent band) of you desired gene (protein) as compared to the protein present in normal cells.

(iii) If you get any gene which is upregulated in glioblastoma cells then you can silence that gene by using siRNA for that particular gene and look for the proliferation of the glioblastoma cells and compare the differentiation with the unsilenced cells. If the result of your RNAsec is true then surely after silencing your gene of interest you will get less proliferation as compared to control cells (as the cancerous cells are known for uncontrolled proliferation).

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