You have reviewed the human genomic sequence and decided to study the function o

Question

You have reviewed the human genomic sequence and decided to study the function of an unknown protein-coding gene. In order to start your research, you must obtain the DNA of this gene. The gene is ~5kb and you are planning to clone the DNA in an expression vector to further analyze protein functions and interactions.

You suspect this gene is expressed specifically in liver cells and would like to analyze the amount of gene expression in different tissues. What method would you use to analyze the amount of mRNA expression of this gene in liver cells, skin cells, neural cells, and stomach cells? Explain the methods used for this experiment.

Explanation / Answer

Expression of this partcular gene product in different tissues can be analyzed with real time PCR or RT-qPCR. During real time qPCR, fluroscence molecules measures the amounft of DNA present in specific sample in each cycle of PCR reaction quantitatively. In comparison to this traditional PCR methods only measures the reaction amount at the end of the reaction. To perform the experiment, first, total RNA or mRNA is required to extract from the respective tissue. Next, the RNA is transcribed to produce cDNA with the help for reverse transcriptase enzyme. After this the cDNA will be used as a template to study the mRNA expression in RT-qPCR with the help of primers specific for DNA sequence of gene of inerest. SYBER green used as fluroscence regent is used as reporter to quantify the amount of DNA generated between different tissue for this partcular gene.

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