Question

Biotechniques

You are interested in studying the effects of glucose on Gene X. Your hypothesis is that expression of Gene X will increase/decrease under high glucose conditions. To prove this, design 5 experiments using the following techniques: Reporter assay sensitivity assay EMSA blot In situ hybridization (IHC with probe for RNA) For each experiment, answer the following questions: What does the technique tell you (what does it measure)? How does this information help support your hypothesis (what part of the molecular pathway is it looking at)? What controls will you use (be thorough - there is often more than 1 good control to use)? What would the results look like (draw the gel or blot or slide or intensity of results)?

Answer 1

a) Reporter gene assay is the assay to measure the expression level of gene of interest.

the DNAse sensitivity experiment says about the the cnanges of gene expression is due to chromatin remodeling or not.

EMSA is a very sensitive method to identify the ptotein-DNA interaction.

western blot is a widely used technique to detect the specific or desired protein in the tissue.

In situ hibridization with probe of RNA is used to do to localize a specific RNA in the tissue.

b) these experiments will give information about the the desired gene expresssion is there or not after the glucose exposure.

is the gene expression changes for chromatin remodelling.

EMSA will give information about the binding of Protein and DNA.

western blot experiment will give information about the expression of protein before and after glucose exposure.

the ISH will give the information about the RNA produced from thre gene before and after glucose exposure.

C) the control should be a group without any treatment of glucose.

D) the result should contain the gel of reporter assay, blot results and the intensity of RNA.