plese help with paper - strange results with MCF7 and GADD45A (Sep/04/2008 )
Hey there. I'm looking for any reference paper where both over-expression or sillencing on one gene had the same effect on expression level of other ( probably target) gene. No metter what I will do to my gene knocdown or overexpression I can see huge increased in mRN of the gene which could be a target. How is it possible, hmm? I'm asking for any paper which will give me the basic ide what is going on. Thnk you for help.
-laska-
QUOTE (laska @ Sep 4 2008, 01:20 AM)
Hey there. I'm looking for any reference paper where both over-expression or sillencing on one gene had the same effect on expression level of other ( probably target) gene. No metter what I will do to my gene knocdown or overexpression I can see huge increased in mRN of the gene which could be a target. How is it possible, hmm? I'm asking for any paper which will give me the basic ide what is going on. Thnk you for help.
Is it possible that you are treating your samples differently? I had a similar thing happen to me when I did miRNA of a plant gene - I saw increased expression of the target. Later I realized that the difference came because non-transformed plants were seeded on regular media whereas the miRNA plants had been seeded on media + hygromycin (to select transformants). I hadn't expected the hyg to make any difference, but apparently it turns on my gene. When I seeded both strains on regular media, there was no difference (the miRNA didn't work to knock down expression). Is it possible that something like could have happened?
-smu2-
QUOTE (smu2 @ Sep 4 2008, 07:03 AM)
QUOTE (laska @ Sep 4 2008, 01:20 AM)
Hey there. I'm looking for any reference paper where both over-expression or sillencing on one gene had the same effect on expression level of other ( probably target) gene. No metter what I will do to my gene knocdown or overexpression I can see huge increased in mRN of the gene which could be a target. How is it possible, hmm? I'm asking for any paper which will give me the basic ide what is going on. Thnk you for help.
Is it possible that you are treating your samples differently? I had a similar thing happen to me when I did miRNA of a plant gene - I saw increased expression of the target. Later I realized that the difference came because non-transformed plants were seeded on regular media whereas the miRNA plants had been seeded on media + hygromycin (to select transformants). I hadn't expected the hyg to make any difference, but apparently it turns on my gene. When I seeded both strains on regular media, there was no difference (the miRNA didn't work to knock down expression). Is it possible that something like could have happened?
no the cells are treated the same way. Hmm its a bit confusing for me
-laska-
QUOTE (laska @ Sep 4 2008, 01:20 AM)
Hey there. I'm looking for any reference paper where both over-expression or sillencing on one gene had the same effect on expression level of other ( probably target) gene. No metter what I will do to my gene knocdown or overexpression I can see huge increased in mRN of the gene which could be a target. How is it possible, hmm? I'm asking for any paper which will give me the basic ide what is going on. Thnk you for help.
Some basic questions which I'm sure you thought of (assuming here that you're doing RT-PCR): Are you doing PCR using primers outside of your siRNA region so that you don't amplify the siRNA? Are you sure that you're doing the RT-PCR correctly such that what you are seeing is truly cDNA and not genomic DNA?
As for a paper, I tried a search myself when it happened to me and I didn't come up with anything. Sorry
smu
-smu2-
Could it be that in both cases you are stressing the cells and the target gene is a stress responsive gene so it will have increased transcription under the stress of KD or over expression?
-bob1-