real-time slopes - (Jan/12/2006 )
In the last months I started to have Real-Time standard curves (gDNA) with high slopes (higher than 4) which means my reaction efficiencies are really bad. I really don't understand what is going on because I am talking about an optimized reaction (primers, annealing temp. PCR protocol, reagents...) I use all the time with very good results. Visually there is nothing wrong with the curves, I have good amplifications and melting curves and the samples I am testing are good too. The problem appears when I analyse the standard curves and I get low efficiencies and high slopes.
I extracted new gDNA for 3 or 4 times to see if the problem was in the gDNA I was using in the standard curves and it didn't work. I already tried 6 or 7 different standard curves of fresh gDNA. Can you help me? Do you have any suggestion?
The only thing I notice is that now the standard curves dilutions are not so close to each other, I mean the number of cycles between the dilutions has increased. I don't know if this is a sign of what is going on… It can be a pipetman problem...
Can I use these curves even with high slopes since my analysis is rather more qualitative than quantitative? I need to compare expression between different genes and not quantify their absolute expression.
Thank you!!!!!!!!!
Gabri
Hi Gabri-
I'm kinda new to RTPCR, so I won't be able to address some of your questions..
But I thought I'd suggest you verify your pipettes in case it helps. If everything else looks good and your r2 is good but your slope is too high, it is possible you are consistently pipetting excess sample into each serial dilution.
The above info is from this (great) source from ABI:
https://www2.appliedbiosystems.com/support/...e_exp_rtpcr.pdf
Good luck!
P.S., If that link doesn't work, you can go here: https://www2.appliedbiosystems.com/support/apptech/#rt_pcr
Click on: Real-Time PCR / TaqMan® Genomic Assays, and it's the "Performing relative quantitation..." under Applications.
...........I extracted new gDNA for 3 or 4 times to see if the problem was in the gDNA I was using in the standard curves and it didn't work..........
The only thing I notice is that now the standard curves dilutions are not so close to each other, I mean the number of cycles between the dilutions has increased. I don't know if this is a sign of what is going on… It can be a pipetman problem...
Can I use these curves even with high slopes since my analysis is rather more qualitative than quantitative? I need to compare expression between different genes and not quantify their absolute expression.
Thank you!!!!!!!!!
Gabri
Dear Gabri,
Probability 1:
The first thing came in my thought was it might due to the primers don't work good any more. Because you said the distance between the std.curves dilutions became greater. For confirmation of this you can compare the CT values of the std.curves from this QPCR with those from former QPCR, in with the same primers were used and the concentration of the gDNA must the same. If there is a shift to higher CT values in every std.curves dilutions, you do better order neew primers.
Probability 2:
You said the QPCR new didn't work any more on the extracted gDNAs , should this due to something went wrong in the extaction, e.g. the reagents didn't work? This could worse the gDNA quality for QPCR.
For your last Q, since I don't know how is your experimental set up and I haven't seen the plots from the std.curves and the samples, I cannot answer you this.
By the was, if the R is close to 1, even you have a high slope, it indicates good pipetman. With slope you know how efficient (=how good) the amplification is. Don't mix up these.
Is it not possible to buy commerical gDNA? It will save you a lot of time and the quality is stable. (At least, if not, money back!
)I hope this help. Success!
I'm kinda new to RTPCR, so I won't be able to address some of your questions..
But I thought I'd suggest you verify your pipettes in case it helps. If everything else looks good and your r2 is good but your slope is too high, it is possible you are consistently pipetting excess sample into each serial dilution.
The above info is from this (great) source from ABI:
https://www2.appliedbiosystems.com/support/...e_exp_rtpcr.pdf
Good luck!
P.S., If that link doesn't work, you can go here: https://www2.appliedbiosystems.com/support/apptech/#rt_pcr
Click on: Real-Time PCR / TaqMan® Genomic Assays, and it's the "Performing relative quantitation..." under Applications.
Thanks for your tips Soluene!
regarding the pipettes I'm waiting for new ones to test again my standard curves. the ct values are lower than expected so I think my gDNA is more diluted than it should be. I'm going to see all the links you sent me. once again thanks for your help.
gabri
......... standard curves (gDNA) with high slopes (higher than 4) which means my reaction efficiencies are really bad. I really don't understand what is going on because I am talking about an optimized reaction (primers, annealing temp. PCR protocol, reagents...) I use all the time with very good results.............
...........I extracted new gDNA for 3 or 4 times to see if the problem was in the gDNA I was using in the standard curves and it didn't work..........
The only thing I notice is that now the standard curves dilutions are not so close to each other, I mean the number of cycles between the dilutions has increased. I don't know if this is a sign of what is going on… It can be a pipetman problem...
Can I use these curves even with high slopes since my analysis is rather more qualitative than quantitative? I need to compare expression between different genes and not quantify their absolute expression.
Thank you!!!!!!!!!
Gabri
Dear Gabri,
Probability 1:
The first thing came in my thought was it might due to the primers don't work good any more. Because you said the distance between the std.curves dilutions became greater. For confirmation of this you can compare the CT values of the std.curves from this QPCR with those from former QPCR, in with the same primers were used and the concentration of the gDNA must the same. If there is a shift to higher CT values in every std.curves dilutions, you do better order neew primers.
First of all thanks for your suggestions! yes, I also thought about the primers so I decided to test other primers. the slopes were higher too. It strange that suddenly all my primers stopped working. It's not impossible but it's really weird... I can explain you; I 've already performed 2 Rtime analysis with all my genes and I had no problems. Now I stopped on my 3rd analysis. and you know visually my curves are great, my samples are great but the slopes... I compared the ct values with previous experiences and the first is similar but the next ones are higher now, that's why I have higher slopes and lower efficiencies. but yes, I think I should try new primers.
Probability 2:
You said the QPCR new didn't work any more on the extracted gDNAs , should this due to something went wrong in the extaction, e.g. the reagents didn't work? This could worse the gDNA quality for QPCR.
First I extracted gDNA the same way I did before, same qiagen kit and same reagents. it didn't work. then we ordered a new kit (the same we used before) and I used "fresh" reagents. It didn't work. we never purify DNA but I even tried that and again it didn't work.
For your last Q, since I don't know how is your experimental set up and I haven't seen the plots from the std.curves and the samples, I cannot answer you this.
By the was, if the R is close to 1, even you have a high slope, it indicates good pipetman. With slope you know how efficient (=how good) the amplification is. Don't mix up these.
Is it not possible to buy commerical gDNA? It will save you a lot of time and the quality is stable. (At least, if not, money back!
)yes, I have great correlation (close to 1 and sometimes 1). It is like I told you, visually everything is ok but I can't trust my results if I don't have a good efficiency. I liked your last tip, buy gDNA. I never thought about that. Thanks again for your help. I'll keep in touch.
Gabri.
I hope this help. Success!