Testing for gene expression is tricky; one needs to extract mRNA, reverse-transcibe it to cDNA and then run quantitaive (Q) PCR to determine copy numbers, with respect to some housekeeping gene (which is assumed to be equally expressed in healthy as well as diseased cells).
Here's interferon gamma cDNA expression from a healthy lymphocyte; note that the cycle threshold (Ct) is 28th cycle.
However, in a sick subject, IFNG is overexpressed, and determined from 22nd cycle onwards.
Determining difference in expression
To put a number on the mRNA expression between the two samples. one also needs to run a housekeeping gene like glyceraldehyde-3-phosphate dehydrogenase GAPDH. This would produce a table like the following:
| Sample | Ct_IFNG | Ct_GAPDH | Difference in Ct |
|---|
| Healthy | 28 | 22 | 6 |
| Sick | 23 | 21 | 2 |
| Difference of differences in Ct | | | 6-2 = 4 |
Thus, the sick sample has 24 = 16 times more IFNG mRNA expression than healthy. (Remember that PCR cycles produce 2n copies after n cycles).
Is the product pure?
This particular run was carried with SYBR green, a dye which preferentially binds to double stranded DNA. Once we start heating the PCR products, at a certain temparature, the products denature, and SYBR green falls off, causing a drop of fluorescence. The sudden change in fluorescence is plotted in the melting curve.
Note that the y-axis is (negative) derivative of fluorescence, i.e. rate of change; thus the sudden drop is reflected in the peak
The melting curve from both samples show a single peak (pure product) which occur at the same temparature (indicating the same product in both runs).
Thanks: Dr Michael John,Sanskriti Rai, Pankaj Kumar & Uddep Chowdhury.
