I divide the average of the experimental gene Cp or Ct (obtained during a qRT-PCR) by the average of the house keeping gene, this gives you a normalized ratio. Then I do stats on this data and graph it. Let me know if that is unclear. Good luck!
We will keep one house keeping gene and one gene of interest in PCR, then we will compare expression of our gene of interest in comparison with house keeping gene whose expression is considered to be unchanged in disease condition.
I divide the average of the experimental gene Cp or Ct (obtained during a qRT-PCR) by the average of the house keeping gene, this gives you a normalized ratio. Then I do stats on this data and graph it. Let me know if that is unclear. Good luck!
Here is a good overview: http://pathmicro.med.sc.edu/pcr/realtime-home.htm
Cheers, Nadine
The "Bible" protocol you'll find under:
http://www.sp.uconn.edu/~ttc02001/MCB3201/Reading%20List%20II/Comparative%20Real-Time%20PCR.pdf
Better you identify a good reference gene whose expression do not changes in your experimental conditions. Like actin, alpha tublin, GAPDH, or ubiqutin gene. Then subtract the reference Ct from healthy from target Ct from healthy. it will give you the Delta Ct. Subtract the reference detla Ct diseased from target delta Ct diseased. Subtract the detla Ct healty from delta Ct of diseased will give you delta delta Ct. Take 2 power - delta delta Ct. It will be the actual fold change in expression of target gene. Dr Dinesh
Dear Dinesh, you really have to test "reference genes" if they really do not change. Everybody suggest reference genes like achtin, GAPHD and others but often they change more than other genes you test. You have to test all your genes with your different treatments and then use the 2 or 3 genes where the lowest difference exist! These genes are the "reference genes"!!!!!!!!!!!!!!
Dear Nadine, I agree with you. But Everybody's suggestion is not enough In an ideal condition no gene is a good reference. So it is exactly empirical to find a good reference gene for specific experimental conditions. Its better to use 2 or three genes with lowest change.
It is an interesting discussion. Yes, the new recommendation is to use 3 house-keeping genes as the control since you will be surprised to see some treatments will change the abundancy of one of the house-keeping gene. Use the least variable one to calculate the delta Ct (Ct-gene of interdst-Ct-house keeping gene). Some people even recommend to calculate the delta-delta Ct ((delta-Ct of treated)-(delta Ct of non -treated). You can easily find several good review by google it.
- Badges
- Science topic