Housekeeping genes for RT-PCR in soybean - (Feb/27/2008 )

Hi everybody,
I need helps concerning housekeeping genes for RealTime PCR. I’m trying to relatively quantify my interested gene expression in different soybean tissues (roots, hypocotyls, cotyledons, leaves, florescent buds, and suspension cultured cells) by SYBR RealTime PCR. That gene is supposed to be at low expression (10-60 copies/ 500 ng total RNA). I have tested Actin, PTB and Fbox for using as housekeeping genes, but none of these genes stably expressed in all tissues.
Do you known other good housekeeping gene?
In the case that there is no good housekeeping gene, I would like to use the external standard RNA to control efficiencies of reverse reaction and those of RT-PCR. Could you suggest me an external standard RNA? Thanks.

I do gene expression in grasses, and we have tested many HK genes for stability. We have found eEF-1alpha and eIF-4a to be the best of 9 HK genes tested. UBQ5 was also very stable, as was 25S, however using 25S can be problematic if your GOI is expressed at a low level because samples need to be diluted to get accurate readings for the 25S. eEF-1alpha was also found to be stable in rice, as was UBQ5. I'm not sure how they will work in soybean, but perhaps some genes to try.

Good luck!

ubiquitin works well with maize as a control for rt-pcr. you can always standarize the expression of your gene with the expression provided by the hkg you pick.

Hi there:

Instead of emailing the whole sequences to you, I can give you the GenBank accession numbers. My suggestion is to BLAST these sequences and make degenerate primers based on other sequences of these genes as well. I know that there are sequences in the database for maize, rice, wheat and other species (hopefully some dicots as well).

Here are the accession numbers:

eEF-1alpha: EU168438
eIF-4a: EF471868 and EF471869
UBQ5: EF470423


I hope this is helpful to you!