Hi Again Anna

find a link to a stratagene guide which itemises in detail ther salient steps in performing qPCR including the machinations of setting up a standard curve

My own summary of this very detailed background material is provided in the word document 'qPCR general overview...' (attached in my qPCR drop box folder)

Finally Anna with regard to data analysis you can quantitate expression using Delta delta ct principles - which pre supposes a near perfect doubling rate, hence the need to obtain primers from standard curve with efficiencies of >/=0.85 - or pfaffl analysis which actually builds in the average rate of doubling in computing gene expression:

see 'qPCR data analysis principles in my drop box folder for details and distinctions

In truth Anna if your primers demonstrate efficiencies of 80% or over, particulalrly if you are trying to ascertain relative expression and not absolute copy number - then the difference between these two methods is negligible and statistically based on repeat assays where actual expression is calculated from CV(10) effectively nothing and would certainely not impact on relative expression trends

I hope all of the above helps and do not hesitate to contact me if you have any more questions

https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0

https://www.dropbox.com/s/4uaa8mbmvlc2fds/qPCR_General%20over%20view%20with%20Specific%20details.doc?dl=0

https://www.dropbox.com/s/d4zh26drtmjc6do/qPCR%20data%20analysis_principles.pdf?dl=0

10 fold dilutions are standard I believe. Either some incorrect math working, or yes inefficient primers. 

This should hopefully be of some help

http://www.sabiosciences.com/newsletter/PATHWAYS07_PCRprimers.pdf

Hello Anna. I am not at work at the moment but have performed many qPCR reactions and will send you some exact protocols in my qPCR drop box tomorrow plus links to useful sites like the above. In essence yes you do need to perform a standard curve by titration your cDNA versus the primers. You then need to create a standard curve using your real time platforms software. There will be clear instructions on how to do this. One thing you always need to do is perform this reaction in triplicate and specify within your experimental design exactly how you diluted your cDNA; that is 10 fold dilutions or 1:2 serial dilution in triplicate of your cDNA. In essence if you have a lot of RNA and can make many copies as cDNA by reverse transcribing from 1ug of RNA then 10 fold dilution from 1:1 to 1:10,000 @ 10 fold intervals will give you a nice curve generally speaking with a good fit. That is R2 > 0.95. Alternatively if your material is more limited and you are obliged to transcribe from much less RNA - say 50ng to 100ng- then in my experience a standard curve based on a serial dilution from 1:1 to 1:64 is much better in terms of a good curve. That is concordant right triplicates which fit the regression line and ct values of between 15 to 35. Generally if your copy numbers are so low that your ct values exceed 40 the triplicates tend to diverge and the standard curve is poor quality. This can happen with low copy number genes from lots of RNA (~1ug) or higher copy number genes from limited amounts of RNA (~50-100ng). Having generated your curve you need to demonstrate that your amplification efficiency is over 80%. That way you can trust actual gene expression data from your cDNA at optimal dilution which you also select from you standard curve. More to follow including proper SOPs tomorrow when back at work !!

Nice answer Laurence~!

Also, for Anna's equational arsenal: 

Be sure to realize that:

1.) Efficiency of the standard curve = [10(-1/slope)] - 1

2.) Exponential amplification PCR efficiency (EAMP) = 10(-1/slope)

(E.g., if Efficiency of the standard curve is 0.83 or 83%, then EAMP = 1.83)

3.) Also, slope = -1/log10(EAMP)

And remember that it is the EAMP values that are the "E" values used in the Pfaffl Method for fold-change calculations (not the standard curve efficiency values). These simple relationships can be put into Excel so you can have hands on your numbers in that format as well.  When you do your dilution study, be aware that the most concentrated samples can inhibit the PCR and that overly dilute samples can also become meaningless as well (invitation of primer dimers, non-specific amplification, Poisson noise, Monte Carlo effect, etc.).  The "sweet range" of your dilution curves is what you are after, and, as Laurence has stated, when analyzing each individual sample for each target, be sure the [optimal] dilution you test them at falls within the good early range of each target's standard (dilution) curve.

Hi Again Anna

find a link to a stratagene guide which itemises in detail ther salient steps in performing qPCR including the machinations of setting up a standard curve

My own summary of this very detailed background material is provided in the word document 'qPCR general overview...' (attached in my qPCR drop box folder)

Finally Anna with regard to data analysis you can quantitate expression using Delta delta ct principles - which pre supposes a near perfect doubling rate, hence the need to obtain primers from standard curve with efficiencies of >/=0.85 - or pfaffl analysis which actually builds in the average rate of doubling in computing gene expression:

see 'qPCR data analysis principles in my drop box folder for details and distinctions

In truth Anna if your primers demonstrate efficiencies of 80% or over, particulalrly if you are trying to ascertain relative expression and not absolute copy number - then the difference between these two methods is negligible and statistically based on repeat assays where actual expression is calculated from CV(10) effectively nothing and would certainely not impact on relative expression trends

I hope all of the above helps and do not hesitate to contact me if you have any more questions

https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0

https://www.dropbox.com/s/4uaa8mbmvlc2fds/qPCR_General%20over%20view%20with%20Specific%20details.doc?dl=0

https://www.dropbox.com/s/d4zh26drtmjc6do/qPCR%20data%20analysis_principles.pdf?dl=0

By the way Jack. Like wise for your excellent answer (as ever)

Thank you all for your fast and nice answers. I will try what you told me.

http://srvgen.upct.es/efficiency.html

I have one ore question. When the cDNA synthesis is done, (let say 20ul of cDNA at the end) first I add 80ul ddH2O to the cDNA and then I make the dilutions for primer efficiency. Is that correct? Otherwise the amount of cDNA is not enough.