Hi
I want to quantify gene expression of apoptotic genes Bax, Bcl-2, and Caspases after treatment vwith my new chemical agent. I have isolated total RNA and genomic DNA from human ovarian cancer cells. I am unable to get bands for Bax, Bcl-2 and caspases after PCR with both genomic DNA and also with cDNA prepared form total RNA. But everytime I am getting good bands with GAPDH. I have done control experiments with GAPDH and my total RNA is devoid of any contamination of genomic DNA.
I am new to PCR so I am unable to decide where I am doing wrong. Please anyone suggest me possible chances of error.
I have used primers which were already published is that a major cause of error? Please suggest.
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pmaj
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Posted 11 March 2010 - 03:51 PM
Hi there,
I also check for the same genes with the drug that we have made. I do REAL-TIME PCR ( I use the STEPONE PLUS instrument from Applied Biosystems), where I use cDNA prepared previously as the template. As far as the genes go, you have to figure out from the literature, when these genes are expressed. I see a difference in these genes at different time points. For Example, I see much more BAX at 24hrs v/s 12hrs. So you may be looking at these genes at earlier time points, and not be seeing much expression. Also if your compound (chemical) is pro-apoptotic, pro-survival genes such as Bcl-2 should generally be lesser in amount. Caspases will also occur at a later time poinst.So check for these pro-apoptotic genes at different time points.
I have not done RT-PCR, so I do not know how much PCR product you need to see a decent band. But I hope this helps.
Bests
Pmaj
I also check for the same genes with the drug that we have made. I do REAL-TIME PCR ( I use the STEPONE PLUS instrument from Applied Biosystems), where I use cDNA prepared previously as the template. As far as the genes go, you have to figure out from the literature, when these genes are expressed. I see a difference in these genes at different time points. For Example, I see much more BAX at 24hrs v/s 12hrs. So you may be looking at these genes at earlier time points, and not be seeing much expression. Also if your compound (chemical) is pro-apoptotic, pro-survival genes such as Bcl-2 should generally be lesser in amount. Caspases will also occur at a later time poinst.So check for these pro-apoptotic genes at different time points.
I have not done RT-PCR, so I do not know how much PCR product you need to see a decent band. But I hope this helps.
Bests
Pmaj
Kashyap, on Mar 11 2010, 11:02 AM, said:
Hi
I want to quantify gene expression of apoptotic genes Bax, Bcl-2, and Caspases after treatment vwith my new chemical agent. I have isolated total RNA and genomic DNA from human ovarian cancer cells. I am unable to get bands for Bax, Bcl-2 and caspases after PCR with both genomic DNA and also with cDNA prepared form total RNA. But everytime I am getting good bands with GAPDH. I have done control experiments with GAPDH and my total RNA is devoid of any contamination of genomic DNA.
I am new to PCR so I am unable to decide where I am doing wrong. Please anyone suggest me possible chances of error.
I have used primers which were already published is that a major cause of error? Please suggest.
I want to quantify gene expression of apoptotic genes Bax, Bcl-2, and Caspases after treatment vwith my new chemical agent. I have isolated total RNA and genomic DNA from human ovarian cancer cells. I am unable to get bands for Bax, Bcl-2 and caspases after PCR with both genomic DNA and also with cDNA prepared form total RNA. But everytime I am getting good bands with GAPDH. I have done control experiments with GAPDH and my total RNA is devoid of any contamination of genomic DNA.
I am new to PCR so I am unable to decide where I am doing wrong. Please anyone suggest me possible chances of error.
I have used primers which were already published is that a major cause of error? Please suggest.
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
~Judah Folkman
~Judah Folkman
