We are using the ABI Prism 7700 Taqman system for our QPCR. This assures more reliable quantification and reduced workload compared to classic competitive PCR methods (which we still use for some targets). The demands that we make for housekeeping genes are the following: - the expression level should be similar between the control gene and the target gene - expression of the control gene is not effected by therapy, infections, etc... Besides this we try to avoid genes for which many pseudogenes are known (such as beta-actin) and we always place our primers over an exon junction. Concerning your last question: I think it is very important to look at this variation between cell types and stimulation conditions. It is of paramount importance to find a control gene that is stably expressed during all your conditions. Another issue is the over time stability. Not only should the control gene be expressed at the same level in different conditions but it should also remain constant as cells are cultured for different periods of time. I hope this is of some help to you. Kind regards, Joeri At 14:49 05/06/2000 -0700, you wrote: > >Hi, a little off the flow topic (but only a little).... > >we are doing quantitative PCR on very small numbers of sorted cells, >and are looking around for a different housekeeping gene for control >purposes than beta-actin. We need one with an intron so that we can >differentiate cDNA from genomic in our ultrasensitive amplifications. > >For people who are using housekeeping RNA's as controls, I'd like to >get feedback: what gene products do you measure? Do you take >advantage of spanning a splice junction? Have you measured the >variation in different cell types or stimulation conditions? > >Thanks for any info > >mr > ================================================ Joeri Aerts Experimentele Laboratoriumgeneeskunde - Laboratoriumgeneeskunde (Hematologie) CDG 7e VD - Gasthuisberg Herestraat 49 B-3000 Leuven BELGIUM tel.: +32-16347012 fax.: +32-16347042 e-mail: joeri.aerts@uz.kuleuven.ac.be ===============================================
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:55:52 EST