RT-PCR Internal Standard
Posted 28 May 2009 - 12:33 PM
Posted 28 May 2009 - 11:46 PM
What are the control genes you have tested?
Beside rRNA, GAPDH and β-actin, other useful internal standard candidates include:
hypoxanthine phosphoribosyl-transferase (HPRT), acidic ribosomal phosphoprotein P0 (36B4), β-2 microglobulin (β2MG), peptidylprolyl isomerase A(PPIA), histone H2A, TATA box binding protein, and tubulin.
Some less popular candidates include: albumin, ATP synthase 6, eukaryotic translation elongation factor 1γ, glucose 6-phosphate dehydrogenase, β-glucuronidase, phosphoglycerokinase, phospholipase A2, porphobilinogen deaminase (hydroxymethylbilane synthase), ribosome protein L-15/L-13/S26, RNA polymerase II, transferring receptor, ubiquitin C, and U2snRNA. The list can go on and on.
I will suggest try HPRT, 36B4, β2MG.
If you have expression profile generated by microarray or some other person you know have done it, you have another option. Get microarray data and find those genes that do not change. As long as they do not change expression level, they can serve as internal standard and they donít have to be house keeping genes.
Posted 29 May 2009 - 09:44 PM
What do you mean with copy # per fixed amount of RNA? Simply use the different CT values from same amounts of RNA? I don't recommend since your RT efficiency might vary. Better use an external standard (spike-RNA), and quantify your data like that.
Hope that helps,