How to do Per cell RNA concentration? - (Aug/16/2006 )

Hi
I was having problems with reproducibility of my qRTPCR, i was reading in one aticle, that they do per cell quantification of RNA. They use spike RNA, i wont use it because my doent have it, also i have read about isoltalion RNA for 5000 cells, or 10 000 etc.
Please, how do youdo it?
Do you count the cell before doing RNA isolation, or do you put same specif number of cell and then you asume that you have a x number.
thanks for any help.
zaira

well my assumptions are not that accurate, but with a certain cell type, i estimate that 80%confluency in my well (6well plate) means roughly 1million cells.
So that's my assumption.
I used once a FACS for giving me a range of cell number... ( 5000 10k 15 ... till 50k)
i don't think counting cells after detachment is suitable as it's enhacing RNA degradation...