LinRegPCR, a beautiful software developed at the AMC, University of Amsterdam. Easy to use, does not assume the same efficiency for each PCR reaction, can be applied to data coming from any qPCR machine.
Plz see: Ramakers et al., NeuroSci Lett 2003; Ruijter et al., Nucleic Acids Research 2009.
(http://www.hartfaalcentrum.nl/index.php?main=files&fileName=LinRegPCR.zip&description=LinRegPCR:%20qPCR%20data%20analysis&sub=LinRegPCR)
Hmm, I am affraid that there is no such freely available software. According to my experiences there are either in-house developed spreadsheet-based semi-automised solutions in labs (like the one Shou Wang described) or rather complex commercial solutions. There is currently none in between.
Software packages that are part of qPCR instrument software are to my knowledge rather limiting and often lack the flexibility for research use.
This was not intended as a pesimistic reply - perhaps as a challenge to find a good solution.
I think you would need to run dilutions of cDNA templates along with your amplification. AFAIK this is done for absolute quantification - I don't know whether it's done with the comparative Ct method.
if you want to do absolute quantification you can use the software in your qpcr cycler. you just need to program correctly. with the ab7300 or 7500 you can enter the values for your plasmid standards. the software will then compare those ct values with your experimental ones.
if you are doing relative expression then you are going to have a hard time trying to put a real numberfor your cDNA levels. You can use many different methods to analyze your data for relative quant. i suggest extracting your data (delta ct and result values in a csv file) and then using the pfaffl method to analyze data.
the modified pfaffl method will adjust for differences in primer efficiencies. this will help you compare your ct values. you will still need to generate an excel file to get a result: induction vs repression. if you need the spreadsheet send me a message and I will help. my spreadsheet will tell you whether a gene is induced, repressed, or not affected when compared to a " no treatment or control treatment" scenario. good luck.
I think an easy to use program is the step one plus from life technologies ( it is free as long as you have the machine ) p.m. If you need more details Pm I might be able to send te program
LinRegPCR, a beautiful software developed at the AMC, University of Amsterdam. Easy to use, does not assume the same efficiency for each PCR reaction, can be applied to data coming from any qPCR machine.
Plz see: Ramakers et al., NeuroSci Lett 2003; Ruijter et al., Nucleic Acids Research 2009.
(http://www.hartfaalcentrum.nl/index.php?main=files&fileName=LinRegPCR.zip&description=LinRegPCR:%20qPCR%20data%20analysis&sub=LinRegPCR)
I also use LinRegPCR and agree with Milka.
Here you can find information about freely and not freely available software, their pros and contras and download links:
http://www.gene-quantification.de/download.html
You can try to use R-package to calculate efficiency for every reaction without plotting of delutions line. It is console-based soft, bit difficult but I have met with interface version.
Another worthy mention is REST (http://www.gene-quantification.de/download.html#rest-384). It has sophisticated permutation algorithms that take qPCR errors into account when testing for the significance of expression regulation. Further it supports calibration curves for your genes and calculates amplification efficiencies which are used for relative expression calculations according to the Pfaffl method. It was supported by Qiagen and shipped with their RotorGene cyclers, but supports every machine.
The website www.gene-quantification.de Vivian mentioned is a very good resource for every aspect of qPCR.
REST won't work on a mac or on a windows 7-machine. You can however use the qPCR package implemented in R; this will do different simulations with your data where one is simmilar to the REST approach. By using R you also stand more freely when it comes to present your data in different figures etc. "all you need to do" is to export your ct-values to a sorted excel file, calculate the efficiencies for the primer-paris and then use e.g the ratiopar command to check for up-regulations/down-regulations of the gene of interest... Good luck! :)
Anette, you're right, the last time I used it was still on a Win XP machine. I checked it with various settings on my W7-64 and could not get it running...
An update: various publicly available curve analysis methods were thoroughly compared by Ruijter et al and the outcome was published in Methods. 2012 Sep 3
(http://www.ncbi.nlm.nih.gov/pubmed/22975077)
Thank you Milka for that excellent reference!
As it turns out, the LightCycler software does a pretty amazing job and is only bested by the Cy0 approach and on par with the LinRegPCR approach.
What I take as conclusion from that article: If you have a LightCycler 480, use the included software (with efficiency correction), if not, use LinRegPCR or Cy0 (if you can live with 1 day waiting for Cy0 results).
#EDIT:
I just saw that the qpcR package for R which was mentioned by Annette also supports the Cy0 method.
http://downloads.lifetechnologies.com/Utilities/Misc/DataAssist3.01_setup.exe
Hi,
First of all, thanks Milka for the link. I just started using it and it turned out to be a great help. I have a question, though. Almost all my samples passed the quality check parameters(0 as mentioned by the software) with few exceptions (5,6) which I suppose is OK. Now, I have 5 targets and 2 reference genes. After I got the output from the LinRegPCR, how should I proceed further? Should I calculate the (N0_Target/N0_Reference) for each sample with both the reference genes and then take the average for the replicates? or should I calculate the normalization factor( NF) (as the geometric mean of N0 for both the reference genes) for each sample? Please help.
Hi Milka, I just tried the software you recommended, It is absolutely great, Thank you so much.
Now I just have one problem that I cant solve with the software instructions. The software is using different terminology for background and baseline fluorescence than I am used to, and it is said that software automatically corrects for the baseline values and that i should not correct it with the QPCR software.
If that is the case, and I want LinRegPCR software to do the baseline correction which fluorescence data do I need to input in the software RN values or DRN (delta RN) values? I would assume that I need to input RN and do automatic baseline correction in LinRegPCR.
Am I correct???
I suggest to use CloudSuite from Thermofisher. This software is constantly updated and more secure space to store your data. https://www.lifetechnologies.com/us/en/home/cloudsuite-scientific-data-analysis-storage.html
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