Answer to Question #97217 in Genetics for Dalchashi

Question #97217
You are a researcher in the Plant Biodiversity Department. Three types of mango leave isolated from Gunung Ledang, Lembah Pantai and Kota Bharu were sent to your department/laboratory. Suggest the most appropriate method to study the expression levels of steroid-related genes (steroid gene A, steroid gene B, and steroid gene C) in all of the samples. Justify why the suggested method is the best approach.
1
Expert's answer
2019-10-24T09:48:35-0400

 I suggest doing a real-time quantitative polymerase chain reaction (PCR) for studying the expression levels of steroid-related genes in the proposed samples.

First of all the workflow is relatively easy. All work process takes 1-2 days and includes 3 main steps. It can be easily performed in the standard laboratory related to the genetics field.

To be more specific and convincing I’ll describe it step by step.

It all starts with RNA isolation. Depending on the financing and reagents that laboratory already has, several options are available. For example, the RNA isolation kit may be used. Depending on the method, it takes 30 minutes to 2 hours to isolate RNA.

After RNA isolation, mRNA should be converted to cDNA. By doing these, researchers are accessing the amount of expressed gene of interest at that time point.

After cDNA is obtained from all three samples, the real-time qPCR step can be performed. This step requires optimization, however, there is a wide variety of relatively cheap kits and master mixes for real-time PCR available. Regarding the fact that these are the leaves from the same organism, only 3 sets of primers are needed (forward and reverse for each gene) to access the expression level. Of course, the reaction should be done in duplicates or even triplicates. In the end, simple calculation allows the researcher to access relative levels of these genes expression.


This workflow can be used in a situation where we know exact target genes and we have a little number of samples and genes of interest. This is what can be seen in the case: there are only 3 samples and 3 genes of interest.


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