Internal Standards for RT-pCR - (Jun/25/2008 )
Hi ,
I have found out that my internal standard is actually changing according to a micro array experiment done on the same samples.
I have been using 18S before. what are my other options? I know that glusose metabolism is also heavily affected as well as membrane proteins ( from microarray results).
Thanks in advance.
u can try any other housekeeeping gene such as actin.
Thanks for the reply.
yes this will be the logical route i should take, but actin is affected as well, but not in the significant range , yet it is affected. I think the whole "house keeping genes" concept is very fragile since in my experiments we use 3 treatments involved in tissue wasting (protein degradation) and another set of animals are treated with all 3 agents. It seems all HOUSE KEEPING genes are drastically affected with these chemical treatments.
you can use GAPDH and a tissue specific housekeeping gene (see pubmed publications) in combination
be aware that even housekeeping genes can be altered by certain treatments, you should test this before doing large experiments and choose two (or even more) that do not change
Have you maybe considered beta2-microglobulin?
Or 36B4
thanks for the all replies. much much appreciated!
I will go back and see how these genes change in miroarray.
does anyone knows any good article about choosing a internal control for tissue specific studies??
Hi
If you have the oportunity to control for the amount of cells you extract RNA from you could also spike the reaction with an artifical standard like firefly luciferase or what ever you like. If you can't control for the amount of cells you can normalize against the same amount of total RNA used for the RT, but this is a bit vague and less accurat as the RNA proportions between cells may vary.
If you search for another HKG you can have a look here: http://medgen.ugent.be/rtprimerdb/ or http://web.ncifcrf.gov/rtp/gel/primerdb/
Lots of information you can find also here: http://normalisation.gene-quantification.info/
Cheers and good luck!
For Internal Control, we always carry out selection with 4 genes before run all samples
HPRT, 36B4, actin and beta2-microglobulin.
And 4 other as back up PPIA, tublin, histone and GAPDH in case first 4 are not good.
you can order the primers of about 12 HKG (sequences are published), run them with sybr green and analyze with the free geNorm software to find the two most stable HKG and then normalize against them (or even more).