Question

You’re working on a newly discovered gene and are tasked to quantitatively quantify the precise transcript levels of this gene being expressed in a culture of cells. Explain the method you will use and details of how you will perform this.

Answer 1

For measuring the expression of a gene we are going to measure the quantity of its mRNA produced. Since transcription is the first step in geene expression, it is a good step to measure expression. For this we are going to use a Northern Blot. We are going to isolate the RNA from our samples and get it ready to be used. We are going to make an step in purification by running the RNA sample is an electrophoresis gel, this will separate the fragments by size so we can discard all the fragments of no use for this experiment.

We are going to create a suitable probe that matches our gene, the probe is a single stranded DNA sequence that complementary to the targget mRNA from our gene, and this probe has a radiolabel for easy identification and quantification. We are going to incubate our RNA sample obtained from electroforesis with our probe. The probe will bind all our mRNAs of interest in the sample, then we run another gel and we quantify the signal level given by the attached labels under specific electromagnetic radiation. This will tell us how much mRNA is in the sample