very high and inconsistent Cts for the house keeping gene - (Jun/03/2010 )
Please help me, I have been doing RT-PCR for the past six months and i still have problems. During this period i was only able to get decent Cts for the house keeping gene (18) twice but all the other times the Cts for both GAPDH/beta actin have been really high 25 or inconsistent within triplicate samples (20, 24, 29). Obviously i cant use these data to analyze gene expression and am getting frustrated. After extracting RNA using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My lab mate normally gets low and consistent Ct for the house keeping gene using the same reagents. I have been trouble shooting for the past 6 months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance
Hi, what is the source material of the RNA? are you checking the RNA quality somehow (gel, bioanalyzer)? How much cDNA are you using for one reaction? did you run a std curve?
Once, a colleage in my department had a similar problem for a long time and then she noticed that she diluted the cDNA too much.
tea-test on Jun 3 2010, 04:26 AM said:
Once, a colleage in my department had a similar problem for a long time and then she noticed that she diluted the cDNA too much.
Am using a nanodrop to measure the concentration and quality of RNA. From the cDNA reaction i use 2ug of RNA and dilute the cDNA 1:50 or 1:25 for qPCR. RNA is being extracted from HEK 293T cells. The standard curve looks normal.
I use 1 ug (sometimes less) of RNA for cDNA synthesis in a volume of 20 ul and usually add 200 ul afterwards. 1:50 seems like a rather large dilution (or, it would be for me...), so I agree with tea-test that you could try a smaller dilution. Maybe you could compare protocols with your lab mate, and see if they use different RT conditions, or different dilutions etc..
Also, when you say 2.500 ng / ul does this mean 2 and a half ng or 2500 ng? (2.5 ng seems very low, especially if using 2 ug in a reaction!) And are the ratios (260/280 and 260/230) nice for the NanoDrop?
Is it only the housekeeping genes which give you poor replicates, or your gene(s) of interest also?
edit: I've just seen your latest reply, and 1:25 should probably be ok, I guess. Maybe you should check that you're using enough of everything for the RT reaction (dNTPs etc), and that there isn't some limiting factor there?
Lapsang on Jun 3 2010, 04:24 AM said:
Also, when you say 2.500 ng / ul does this mean 2 and a half ng or 2500 ng? (2.5 ng seems very low, especially if using 2 ug in a reaction!) And are the ratios (260/280 and 260/230) nice for the NanoDrop?
Is it only the housekeeping genes which give you poor replicates, or your gene(s) of interest also?
edit: I've just seen your latest reply, and 1:25 should probably be ok, I guess. Maybe you should check that you're using enough of everything for the RT reaction (dNTPs etc), and that there isn't some limiting factor there?
Thanks
The 260/280 and 260/230 ratios looks good and i meant 2500ng of RNA. My lab mate and i use the same protocol and reagents and it works for him but he is been doing it for a very long time now. The mechanistic part of the experiment is the only thing that is different, could i be over drying or underdrying my samples after ethanol wash, how big of a difference can pipetting errors or vortexing and a quick spin after each and every step make? Could it be that my pellet is not completely resuspended when i OD?