CT value>25 with GAPDH in 50-fold dilution! - (Mar/16/2011 )
I have got consistently ct value>25 with GAPDH in qPCR. The cDNA were come from 2ug of RNA of cell lines like u87, A172, u373 and etc. Then diluted to 50 folds. I have changed the sybr green with other brand. The result was unchanged. Is there anything wrong? Please help. I am desparate.
Have any of you encounter this high ct in housekeeping? Please share your experience with me.
If >25 means like 25-29, it's perfectly OK value, there are low and high abundancies in housekeeping genes (though GAPDH isn't exactly low abundant), because ideally it should have similar Cts as your target genes. Why are you such desperate, you need to dilute it further?
50x is quite a big dilution, I use 1 ug of RNA to 20 ul RT reaction, don't dilute it, usualy use 2 ul and get Cts as low as 17 or as high as 23. Maybe your primers are not that efficient, did you calculate the efficiency? If it's going to be around 90 - 110% you have no problem, maybe there is just not that enough GAPDH in your RNA.
Thanks, veteran. I am desperate because I think I have something wrong which I have spent lots of time. I am so green.