Question

Beginning with 1 g of liver tissue from both a rat treated with glucagon and a rat left untreated (control), describe two methods that would allow you to estimate the change in level of the mRNA for the enzyme, glycogen synthase, upon glucagon stimulation. Please give as much detailed answer as possible, I'm really trying to understand the concept, thanks.

Answer 1

Even though nearly every cell in an organism's body contains the same set of genes, only a fraction of these genes are used in any given cell at any given time. It is this carefully controlled pattern of what is called "gene expression" that makes a liver cell different from a muscle cell, and a healthy cell different from a cancer cell.Gene expression is dynamic, and the same gene may act in different ways under different circumstances.

In order to activate a gene, a cell must first copy the DNA sequence of that gene into a piece of mRNA known as a transcript. Thus, by determining which mRNA transcripts are present in a cell,it can determined which genes are expressed in that cell at different stages of development and under different environmental conditions.

The quantity of mRNA transcript for a single gene directly reflects how much transcription of that gene has occurred. Tracking of that quantity will therefore indicate how vigorously a gene is transcribed, or expressed. To visualize differences in the quantity of mRNA produced by different groups of cells or at different times, researchers often use the method known as a Northern blot. For this method, researchers must first isolate mRNA from a biological sample by exposing the cells within it to a protease, which is an enzyme that breaks down cell membranes and releases the genetic material in the cells. Next, the mRNA is separated from the DNA, proteins, lipids, and other cellular contents. The different fragments of mRNA are then separated from one another via gel electrophoresis.and transferred to a filter or other solid support using a technique known as blotting. To identify the mRNA transcripts produced by a particular gene, the researchers next incubate the sample with a short piece of single-stranded RNA or DNA (also known as a probe) that is labeled with a radioactive molecule. Designed to be complementary to mRNA from the gene of interest, the probe will bind to this sequence. Later, when the filter is placed against X-ray film, the radioactivity in the probe will expose the film, thereby making marks on it. The intensity of the resulting marks, called bands, can tell us how much mRNA was in the sample, which is a direct indicator of how strongly the gene of interest is expressed