PCR Inhibitor - Where/What are you? (Dec/19/2008 )
So I am having an issue of moderate PCR inhibition with Syber Green. I think this is the case because I am getting slopes of -2.8 to -3.0, which would suggest a 120-130% efficiency. When I look at my standard curve closer the efficiency improves the lower on the curve I go, suggesting a dilution of inhibitor, rather than improving with removal of the low points (which would suggest primer dimers). Now my RNA was of good quality, 2-2.1 A260/280 and I did not have ethanol carryover from my Qiagen columns A260/230 > 2.0. I use just RNA that was isolated at the same time from the same tissue type to generate all of my standard curves. Previous standard curves for both gDNA, mtDNA, and RNA isolated at a different time have appropriate efficiencies 90-110%. How do you recommend troubleshooting this? Use low concentration template if ct is less than 35? Reisolate RNA? Resynthesize cDNA?
my best suggition is you have to isolate RNA again
An update on the situation. I ran some more standard curves. Same standard, same water used for the dilutions, same water used for the primer dilution, and I got one curve at -3.2 (-3.4 without the highest sample) and then the other curve was -3.45.
That being said I think I am going to try running another curve with a blend of my real samples to see if by chance it was just that one sample i have being using as a standard.
An update on the situation. I ran some more standard curves. Same standard, same water used for the dilutions, same water used for the primer dilution, and I got one curve at -3.2 (-3.4 without the highest sample) and then the other curve was -3.45.
That being said I think I am going to try running another curve with a blend of my real samples to see if by chance it was just that one sample i have being using as a standard.
Have you done a standard PCR on your cDNA -RT to be sure you do not have genomic DNA contamination and to check the levels of primer dimers?
You can also use the Pfaffl method to analyze your data as it takes into account your varying efficiencies.
You can also use the Pfaffl method to analyze your data as it takes into account your varying efficiencies.
I have not checked for gDNA contamination (although I did an DNAase step), but I did check for primer dimers, which I do not have. Can you use the Pfaffl method to look at efficiencies above 110%? I thought it was only for <100% efficiencies.
Have you cleaned up your RNA stock?
Last time we also have similar problem and finally we realized it is inhibitor in RNA stock.
After we try LiCl clean up before DNase, we got a very good titration curve. (from -2.28 to -3.24)
I think our inhibitor is carbohydrate and that will not affect OD reading that we usually test, so it is very easy to miss.
Last time we also have similar problem and finally we realized it is inhibitor in RNA stock.
After we try LiCl clean up before DNase, we got a very good titration curve. (from -2.28 to -3.24)
I think our inhibitor is carbohydrate and that will not affect OD reading that we usually test, so it is very easy to miss.
Do you think that the order of clean-up and DNase treatment makes a difference? We use columns to clean-up w/DNase digestion on-column.
I don't think the order will make any difference.
Because of our RNA extraction procedure, we do clean up before DNase treatment.
Check with company, if your columns also remove carbohydrate and lipid then I am useless for this topic.
Has anyone tried this method?
http://www.gene-quantification.de/nolan-spud-2006.pdf
Cheers
Chris