Variation i the referance gene expression

[sampada]

Hi,'
I am working with the A549 cells and I used the referance gene panel to find out the appropriate ref gene .GeNorm shows that GAPDH is the best one. But when it shows differant expression as the treatment of the cells changes.????Can anyone suggest what can do or is this normal to have some differnce in the expression of ref gene as the treatment to the cells changes?????

I hope somebody can help.

Thank you
Sampada

[Mikael Kubista]

Sampada,
geNorm is not the best method to identify best reference genes if you are comparing two groups. You should use the somewhat more advance Normfinder. Normfinder differentiates between expression stability and differential expression between groups. Normfinder is available in, for example GenEx (www.multid.se).

Good luck

[sampada]

Hi Mikael,
Thanks a lot for your reply.
I have also tried using NormFinder . It also shows that GAPDH is the best gene. I used the REFERANCE GENE PANEL from TATA Bioscience. Could there be a difference in the seq of GAPDH provided by TATA Bioscience in the kit and the one which I have.?? Could this be a reason for variation?? If yes then can I get the same GAPDH primers from TATA Bioscience??

Also is the diffence of cycle in treated and untreated samples ok to say that the referance gene is working fine??.

Thank you.

Sampada

[Mikael Kubista]

Usually it dis not a problem to change assay assuming the new assay is as sensitive and robust. Theoretically, there may be problem if the assays detect different splice variants or different pseudogenes, but we never encountered this, at least not yet. In any case, if you found the GAPDH assay in the TATAA nabel to give you highly stable readings save yourself time and purchase just that single assay. You can order it here:

human: http://www.tataa.com/products/Individua ... ducts.html

mouse: http://www.tataa.com/products/Individua ... ducts.html

If data are normalized there should not be a difference between treated and untreated samples for reference genes. But normalization must be appropriate. Be aware, normalization to total amount of RNA is not always acceptable (but it can be tested with Normfinder).

Good luck

[sampada]

Hi Mikael,
thank you for your last reply.
Can you tell me in detail how can I normalize my data???
I have a dose response curve with a control that is not treated and the rest are treated with increasing concentrations of diffent enzymes.
I stimulate the cells and isolate the RNA followed by preparation of cDNA and then run the qPCR for target gene(IL-8).
Please tell me how to normalize my data in order to get constant or stable expressio n of referance gene.

Kind regards,
Sampada Bhagwat

[Mikael Kubista]

Hi Sampada,
We can help you analyze your data through our priority customer (P3) program. Contact me on mikael.kubista@tataa.com and I wille xplain how it works.

Regards
Mikael