Hi Nurul,

The Livak-Scmittgen equation addresses this as well:

http://dingo.ucsf.edu/twiki/pub/Cores/BioinformaticsCore/QPCR/Livak_Schmittgen2001.pdf

It is the 100% Efficiency version of the Pfaffl Ratio Method in the Chapter 3 I sent.

Also see the attached Excel file.

The trick is knowing in which order to subtract the Ct values in the exponent part of the equation. If you subtract Control Ct from Treated Ct or if you subtract Treated Ct from Control Ct; you get the reciprocal result. One is considered fold down regulation, while its inverse is considered up-regulation. It's all a matter of being in control of and aware of which way you flip the fraction (e.g., which way you subtract the exponent Ct values; since when you log values that are subtracted, that implies division by mathematic principle) ... likewise when you add exponents, that implies multiplication by basic log rules as well.

See the attached Excel file for clarification.

You are performing a "delta Ct" method when normalizing to a reference gene.

And, when you divide this result by a control ("calibrator") sample target level already normalized to its respective GA3PDH level, then you are performing a "delta delta Ct method". E.g. 2 to the -delta delta Ct or the efficiency corrected Pfaffl Ratio Method (which are identical methods when all Eamp values = 2 (e.g. all target and ref genes are assumed to amplify at 100% efficiency). See attached.

Where exactely is your problem?

http://www.ncbi.nlm.nih.gov/pubmed/11846609

You can find a link to a free PDF of this article when you google for "Livak_Schmittgen2001.pdf‎" under dingo.ucsf.edu/twiki/.../Livak_Schmittgen2001.pdf‎.

See also:

https://www.researchgate.net/post/Statistical_analyses_with_fold_change_significance_values

https://www.researchgate.net/post/How_to_analyze_a_qPCR_with_2_or_3_housekeeping_genes

https://www.researchgate.net/post/How_can_I_estimate_statistical_differences_between_calibrator_and_test_conditions_in_Relative_Quantification_qPCR

You may want to use the comparative Ct method, it´s easy to understand. I recommend you to download "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR", of Applied Biosystems. In section VII, you have everything you need to start doing your calculations for relative quantitation.

Part 1 of Bio-Rad Video http://www.youtube.com/watch?annotation_id=annotation_865814&feature=iv&src_vid=tgp4bbnj-ng&v=GQOnX1-SUrI

Part 2 http://www.youtube.com/watch?annotation_id=annotation_741628&feature=iv&src_vid=GQOnX1-SUrI&v=tgp4bbnj-ng

Easy and comprehensive step by step explanation

Q Base is a good programme to analyse qPCR data. It does all the calculations based on your housekeeping/control genes

control: 18/25=0,72

test: 20/23= 0,87

If control is 1:

1:0,72=x:0,87

x=1,208

1,208 is the fold change of gene x in test condition in comparison to value 1 of control condition.

Federico, no. The values given are very likely ct values, not concentrations!

In that case, Jochen, you are right. Thus, I suggest to do simply the same calculations with the concentrations values of gene X and GAPDH to obtain the fold change.

If you are assuming perfect efficiencies for both your GA3PDH and your gene of interest, the simple calculation would be:

[2^(18-20)] / [2^(25-23)] which = 0.0625

Meaning that your gene of interest has 16-fold less expression in your test condition as compared to your control condition... (1/0.0625 = 16).

The "2's" in the above equation are your base of exponential amplification "Eamp" values indicating 100% efficiency.

In most real situations, efficiency is not 100% (so look out!)....

Recall that Eamp = 10^(-1/slope) or 2^(-1/slope) if using log base 2.

and

Efficiency (E) = Eamp - 1

Hi Nurul,

The Livak-Scmittgen equation addresses this as well:

http://dingo.ucsf.edu/twiki/pub/Cores/BioinformaticsCore/QPCR/Livak_Schmittgen2001.pdf

It is the 100% Efficiency version of the Pfaffl Ratio Method in the Chapter 3 I sent.

Also see the attached Excel file.

The trick is knowing in which order to subtract the Ct values in the exponent part of the equation. If you subtract Control Ct from Treated Ct or if you subtract Treated Ct from Control Ct; you get the reciprocal result. One is considered fold down regulation, while its inverse is considered up-regulation. It's all a matter of being in control of and aware of which way you flip the fraction (e.g., which way you subtract the exponent Ct values; since when you log values that are subtracted, that implies division by mathematic principle) ... likewise when you add exponents, that implies multiplication by basic log rules as well.

See the attached Excel file for clarification.

You are performing a "delta Ct" method when normalizing to a reference gene.

And, when you divide this result by a control ("calibrator") sample target level already normalized to its respective GA3PDH level, then you are performing a "delta delta Ct method". E.g. 2 to the -delta delta Ct or the efficiency corrected Pfaffl Ratio Method (which are identical methods when all Eamp values = 2 (e.g. all target and ref genes are assumed to amplify at 100% efficiency). See attached.

(Mean-Q x V1 x ρ1) /M x ρ2

Mean-Q: the Value form the original data.

V1: The volume of DNA when finished your DNA extraction for Xg sediment.

ρ1: The DNA concentration of V1.

M: The gram of sediment used to extract DNA.

ρ2: The DNA concentration you used to do Q-PCR.

what is the unit we can use for this formula

http://www.sabiosciences.com/manuals/seminars/qPCR%20Introduction_April%202012.pdf