Question

A graduate student decided to investigate the effects of heat on the expression of the VirD gene of Agrobacterium tumefaciens cells. He grew two batches of cells, one at 28 ^oC and the other at 32 ^oC. He then measured the transcriptional expression of the VirD gene by qPCR in both cells, and collected the following data.

	Average CT (VirD); n = 3
Grown at 28 oC	25
Grown at 32 oC	20

a) What does this data imply about the relative expression of the VirD gene grown at different temperatures?

b) Is the experiment reliable ? Explain?

Answer 1

In real time PCR analysis as CT value for a particular gene increases in a sample it explains that the expression of that gene is less in that sample compared to the other sample . In this experiment, the CT value for VIRd gene is more for cells grown at 28 degree Celsius compared to cells grown at 33 degree Celsius . This demonstrates that the expression of VIRd gene is more in cells grown at higher temperature. You will understand it more if we go in detail about the principle of real time PCR using syber green. The syber green used in real time PCR, will get incorporated in to double stranded nucleotides. Once incorporated in to double strand the fluorescence emitted by it will be increased. In the real time PCR, a threshold fluorescence is taken as the one CT value. That is the cycle of amplification required to reach the threshold CT value will be less if the particular gene is highly expressed in the sample .

But the above experiment is not a reliable one. The reason is that they have not analysed the expression of an house keeping gene. The expression of VIRD gene should have been normalised to a house keeping gene. House keeping gene is always included to check the quality and quantity of mRNA, and to check if there is any problems with the various enzymes used during the experiment, pipetting errors etc. Examples for house keeping gene bacteria are 16s RNA,gmk, DHFR gene etc.