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very high and inconsistent Cts for the house keeping gene - (Jun/02/2010 )

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

-gradechips-

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-

tea-test on Jun 3 2010, 04:26 AM said:

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.


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.

-gradechips-

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-

Lapsang on Jun 3 2010, 04:24 AM said:

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?

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?

-gradechips-