Hi all, Anyone have a good reference for how to normalize and determine significance of fold change for RT-PCR data? Basically, we have 6 control individuals and 6 experimentals. We normalize using 18s, but our RNA measurements going into the machine are pretty noisy, so we need to know what people who **successfully** confirm microarray data with RT-PCR are up to... TIA, Ken

Hi Ken, 18S rRNA is a good starting point because it is stable, but the disadvantage is that the expression level is very high. Ideally, the housekeeping gene and target gene should be expressed at a similar level, therefore you should target a stable mRNA rather than 18S rRNA. I followed the literature below to get a stable gene for human T-cells Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics. 2000 Apr 27;2(3):143-7. PMID: 11015593 [PubMed - indexed for MEDLINE] Hamalainen HK, Tubman JC, Vikman S, Kyrola T, Ylikoski E, Warrington JA, Lahesmaa R. Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Anal Biochem. 2001 Dec 1;299(1):63-70. PMID: 11726185 [PubMed - indexed for MEDLINE] The following website also has everything you need to know about real time PCR including house keeping gene section, validation of microarray, software etc........ http://qPCR.gene-quantification.info/ regards Anthony >Hi all, > >Anyone have a good reference for how to normalize and determine >significance of fold change for RT-PCR data? > >Basically, we have 6 control individuals and 6 experimentals. We >normalize using 18s, but our RNA measurements going into the machine >are pretty noisy, so we need to know what people who >**successfully** confirm microarray data with RT-PCR are up to... > >TIA, >Ken > >_______________________________________________ >Bioconductor mailing list >Bioconductor@stat.math.ethz.ch >https://stat.ethz.ch/mailman/listinfo/bioconductor -- ______________________________________________ Anthony Bosco - PhD Student Institute for Child Health Research (Company Limited by Guarantee ACN 009 278 755) Subiaco, Western Australia, 6008 Ph 61 8 9489 , Fax 61 8 9489 7700 email anthonyb@ichr.uwa.edu.au