Posted 20 March 2011 - 10:28 AM
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.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.