Hi Amin,

As you maybe aware, there are numerous houskeeping genes that can be used for real-time PCR (beta-actin, GAPDH, 18S, YWHAZ, alpha-tubilin - just to name a few!).

However, it is best to determine which housekeeping gene is best for the particular cells/tissue you are working with (refer to previous studies if already published). Otherwise, you will need to first establish constant (non-variable) expression of one (or more) housekeeping gene before continuing with your experiments.

We certainly had this issue in human placenta, hence our group published the following paper which may be of use to you:

Article: GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.

Authors: P Murthi, E Fitzpatrick, A J Borg, S Donath, S P Brennecke, B Kalionis

Jounal:  Placenta 10/2008; 29(9):798-801.

All the best,

Gayathri

A few more comments:

• Beta-actin usually has a good and stable expression, but due to short introns, it is difficult to get primers that discriminate well against gDNA. Maybe you can make some that span the boundary between two exons.
• Ideally, the expression level of the housekeeper is close to expression level of the gene of interest. If expression of 14-3-3-zeta (YWHAZ) is stable in the thymus, this might fit this criterion better than the classical housekeeper genes.

Hi Armin,

Please be careful regarding pseudogenes. Since the attached paper appeared we are using HPRT as reference gene.

Best,

Jochen

Good luck!

Cheers,

Jochen