You also probobably need to check if your samples haven't been contaminated with PCR reaction inhibitors, which is very common if you first extract your mRNA, digest remaining DNA and then run a PCR with a less then 10-fold dilution. You need at least a 200 times dilution to get rid of all the artifacts.
Once you've diluted your samples, place a repeats of successive dilutions of your target and baseline mRNA (200xdilution, 1000x dilution, 5000x dilution). Normally you will see a nice regular spacing between curves on your rtPCR plot. If you don't, something went wrong with the reaction.
More generally rtPCR is a pretty tricky experience, and if you want it to be 100% reliable, you've got a certain number of controls to perform. You can find a good and comprehensive guideline over here.
No comments:
Post a Comment