Problem - same ct values in my RT positive and negative samples - (Nov/10/2006 )
Hi,
I could really use some help on this one. I've recently been getting the same values in both my postive and negative samples for several genes. They are both coming up at around 29-32. I thought this was obviously a contamination, but I've changed all of my reagents, set up the plate in another area of the lab, cleaned my pipettes throughly (with a solution designed to remove RNase and DNA contamination) and used someone else's pipettes, got someone else to make up new primer and probe solutions from reordered stocks, and used cDNA that I have had undetectable results for my GOI before, and used someone else's cDNA. And still I get these Ct values. I have run several other genes and they come up undetectable when they should be.
The genes that give me the weird ct values, I have recently being trying to clone, so that's why my first instinct was contamination with plasmid. But, two of the genes that I get these weird ct values I haven't been able to get yet, and I taken every precaution I can think of to elimate it.
Has anyone else every encountered something like this before? If so how did you resolve it? I've been at this for about 3 weeks and I am getting extremely frustrated with it.
Any tips on where else the contamination could be hanging on from?
Thanks in advance.
FecX - PhD Student in Hell
I could really use some help on this one. I've recently been getting the same values in both my postive and negative samples for several genes. They are both coming up at around 29-32. I thought this was obviously a contamination, but I've changed all of my reagents, set up the plate in another area of the lab, cleaned my pipettes throughly (with a solution designed to remove RNase and DNA contamination) and used someone else's pipettes, got someone else to make up new primer and probe solutions from reordered stocks, and used cDNA that I have had undetectable results for my GOI before, and used someone else's cDNA. And still I get these Ct values. I have run several other genes and they come up undetectable when they should be.
The genes that give me the weird ct values, I have recently being trying to clone, so that's why my first instinct was contamination with plasmid. But, two of the genes that I get these weird ct values I haven't been able to get yet, and I taken every precaution I can think of to elimate it.
Has anyone else every encountered something like this before? If so how did you resolve it? I've been at this for about 3 weeks and I am getting extremely frustrated with it.
Any tips on where else the contamination could be hanging on from?
Thanks in advance.
FecX - PhD Student in Hell
Have you looked at the dissociation curves and/or run the product out to check the length of the fragment?
When my negatives give ct values, they are always primer dimers but it isn't obvious without checking the dissociation temp and/or product on a gel.
I agree with Patty it could be because of primer dimers. Check the dissociation curve of the negative controls and run positive and negative on a gel
Hi,
Thanks for the input, it is much appreciated. Touch wood I have found where the problem was - a contamination in a component of the RT mix that I was using to make my cDNA. I tried new RT MIX (from new unopened vials of enzymes, buffers, etc) and it seems to have done the trick.
Cheers,
FecX