real time pcr newbie - (Jun/23/2008 )
hey guys, i am just starting to learn real time pcr. i am trying to quantitate the levels of two different enzymes in a murine cell line. i have never used real time pcr before and at this point, i do not even have a protocol for this yet.
specifically, i would like to know how far apart should the spacer between the forward and reverse primers be? also, what is the nature of the fluorophore - is it a dye or it is a complementary piece of DNA (with a flurophore) to the intervening region between your primers? additionally, what is best used as a reference gene?
basically, any insights / experiences that you wish you knew when you first started real time pcr that you would like to share would be very helpful. thanks very much.
specifically, i would like to know how far apart should the spacer between the forward and reverse primers be? also, what is the nature of the fluorophore - is it a dye or it is a complementary piece of DNA (with a flurophore) to the intervening region between your primers? additionally, what is best used as a reference gene?
basically, any insights / experiences that you wish you knew when you first started real time pcr that you would like to share would be very helpful. thanks very much.
Check out this introductory slide show, hope it helps: www.dorak.info/genetics/realtime.ppt
Good luck!
A number of introducory documents could be downloaded from the Applied Biosystems web-page.
Hi,
unfortunately there is no general housekeeping gene. The reference gene you use depends on the experiment you are running and the organism and the tissue you are working with. Anyway a careful validation of you HKG is recommended nowadays proofing that it is expressed stable and not affected by experiments you are doing. Infos and reference see here http://normalisation.gene-quantification.info/
Cheers