Question

Objective 1:

You run a transcriptome analysis and find that Lis1 is a gene that is has a loss-of-function mutation more often in ASD patients compared to non-ASD controls. You would like to understand how this mutation manifests in a rodent model.

1. Please describe what ‘loss-of-function’ means.

              Loss-of-function means that this Lis1 gene’s product (protein) will have less to no function at all. With less function, the gene product will only be partially inactivated, but with complete loss of function it is amorphic.

2. Please describe how you can examine the effects of decreased Lis1 function in mice at the RNA and protein levels.

             

I need help with the 2nd portion of this question. She is basically asking how you would measure changes in RNA and changes in protein, but I am not sure what the best method would be to do either one of these. Qpcr?

Answer 1

To examine the effects of decreased lis1 function in mice at RNA level, we can use the following techniques-

1. Northern Blotting- It shows the size and sequences of the used mRNA molecules. A sample of RNA is separated on an agarose gel and is allowed to be hybridised with a radiolabelled probe and is detected by autoradiography.

2. reverse transcription- qPCR- A DNA template called cDNA is generated using a mRNA template and the DNA is then amplified along with a labelled probe which emits flouroscence. It shows the exact copy number of the original mRNA.

3. For high thoroughput analysis, other techniques such as hybridisation microarray and next generation sequencing can be done but can be expensive.

To measure changes in protein level, we can use the following techniques -

1. Western blotting- Size of the protein of interest can be identified through this technique. First, protrin samples are seperated by SDS PAGE and then transferred onto membrane and probed with antibody for detection. Lastly it is conjugated with a flourophore for quantification.