LinReg calculations - Does anybody have experience using LinReg with Light Cycler data? (Feb/03/2006 )
I started using Q-RT-PCR a year ago. We were using the Light Cycler Real Quant program for quantitative calculations. Then we became aware of LinReg, a program that could be use to calculate PCR efficiencies in each reaction.
Using the LinReg program to calculate PCR efficiency of many runs with different primers gave me surprising results:
If I use LinReg, the PCR efficiencies are under 1.7, sometimes down to 1.4
If I use a dilution curve for primer efficiency calculations, then the same primers give an efficiency of 1.85 to 2!
For the LinReg calculations I am exporting the data from the lower graf in the Light Cycler, is that right? or should I use a different file?
For a comparisson, I saw calculations made from data derived from a different Q-PCR machine, and their figures for efficiencies with LinReg were much closer to 2. I do not believe that all our many primer sets are so much worse than all the primer sets they were using, they were all designed based on the same rules and with similar programs.
Well, I hope somebody can help,
thank you
Ccv
We also tried to use LinRegPCR. And we had the same problems with samples, that e.g. had a high fluorescent background from genomic DNA. There's no real conclusion, why it didn't work, but we decided, that the data the program uses, is already beyond the turning point in the PCR, when the efficiency has begun to decline. LinRegPCR seems to work with some samples. But other samples, although it is proven, that their cp-values aren't inhibited, can't be correctly evaluated by this program.
Thank you very much for answering. Now, that we are in the subject, I want to share that we are trying to find out which are the best method of quantifying the results from the Light Cycler. It seems that RealQuant does not take enough aspects of the PCR into consideration and the results do not always seem reliavable. Is it possible for you (or other reathers) to share experiences about Real time quantification:
1- The use of several standard genes
2- Use of RealQuant
3- Use of REST or other quantification software
4- Which kind of crosspoint do you find best: 2nd derivative or fit points
5- Do you know any other program that will do a better job than LinReg to calculate the PCR efficiency in individual reactions or do you use primer efficiency based on dilution curves?
Well, I know that there are many questions, but that is what we are dealing with when we want to do the things "RIGHT??" so I will appreciate any indput with suggestions from people that have been through similar or the same questions, and I will be particularly interested in knowing if anybody have reach any conclussion of how to do this calculations and why.
I will post this questions also as a new subject to try to reach more readers
Thank you again
CCV