[BioC] RT-PCR normalization & stats reference

[Anthony Bosco]

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
>
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-- 
______________________________________________

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 at ichr.uwa.edu.au