Non repeatability of RT-PCR with same cDNA - (May/07/2007 )
hi to all,
I am doing an assay in which RT-PCR is to be done with RNA samples taken at different time points under treatment of a drug from the same plate.
I am not able to get same profiles for the same cDNA series.
What do you think has gone wrong?
There are two inputs from my side.First there was variation in the volume of PCR reaction mix after the program ended, even when heated lid was used. Thus I started adding mineral oil and tested all the wells for the same cDNA and primer set. all the wells gave similar results. Next I store cDNA at -20, during storage I have seen precipitate in all the tubes, generally I vortex the tube and then take the cDNA but even on vortexing it does not go away.For making cDNA I use HSRT kit from sigma.I have been at this since a month and till now I have not been able to get repeatable results other than when I was testing the different wells with same cDNA and same master mix in which cDNA was also added.
Till now I have reached the conclusion that machine is fine and so are the chemicals. Does anyone have suggestions?
thanks
mili 
I am doing an assay in which RT-PCR is to be done with RNA samples taken at different time points under treatment of a drug from the same plate.
I am not able to get same profiles for the same cDNA series.
What do you think has gone wrong?
There are two inputs from my side.First there was variation in the volume of PCR reaction mix after the program ended, even when heated lid was used. Thus I started adding mineral oil and tested all the wells for the same cDNA and primer set. all the wells gave similar results. Next I store cDNA at -20, during storage I have seen precipitate in all the tubes, generally I vortex the tube and then take the cDNA but even on vortexing it does not go away.For making cDNA I use HSRT kit from sigma.I have been at this since a month and till now I have not been able to get repeatable results other than when I was testing the different wells with same cDNA and same master mix in which cDNA was also added.
Till now I have reached the conclusion that machine is fine and so are the chemicals. Does anyone have suggestions?
thanks
mili
you haven't written detail of your problem. wht sample you are using for RNA extraction, wht mehod you are using for RNA extraction and cDNA synthesis. after that one can comment on the problem.
Hi,
I am using RNA from K562 cells, method for extraction is trizol, I use TRI reagent (Sigma).For cDNA synthesis I use HSRT kit 100 Sigma.
mili
try with same cDNA from single source (Well) and set three different PCR reaction and see wheather u r getting the same profile or not. If not there is problem in quantification of RNA used for cDNA synthesis or in pipetting.
first standerdize the reaction with untreated K562 cell lines.
otherwise no problem seems in the detailes provided by you.
All the Best.