I need to know the form to analize and make charts of relative quantification of differents cytokines mRNA expression, but using the deltaRn, not the Ct provide by the software. I'm using Applied Biosystems StepOne with Sybr green reagents.
Applied biosystems has a quite analytical guide for calculating DeltaCt and DDCt for comparative quantification of samples. Very briefly to calculate the DCt of each sample you need to subtract the Ct value of the endogenous reference gene in that sample (e.g GAPDH) from the Ct value of the target gene (your cytokines) in that sample. Then to compare two or more different sample you need to calculate the DDCt. This is by subtracting the DCt of the ''control sample'' (for example untreated cells, or wild type tissue) from the DCt of the experimental sample (e.g treated cells, or mutant tissue). Then the fold change of the target gene in that sample is calculated with the formula: 2 power(–∆∆CT).
Here is a link to Applied Biosystems guide:
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf
Hope this is helpful!
Applied biosystems has a quite analytical guide for calculating DeltaCt and DDCt for comparative quantification of samples. Very briefly to calculate the DCt of each sample you need to subtract the Ct value of the endogenous reference gene in that sample (e.g GAPDH) from the Ct value of the target gene (your cytokines) in that sample. Then to compare two or more different sample you need to calculate the DDCt. This is by subtracting the DCt of the ''control sample'' (for example untreated cells, or wild type tissue) from the DCt of the experimental sample (e.g treated cells, or mutant tissue). Then the fold change of the target gene in that sample is calculated with the formula: 2 power(–∆∆CT).
Here is a link to Applied Biosystems guide:
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf
Hope this is helpful!
The Cy0 method most likely is what you are looking for here - as it relies on the 5-parameter Richards equation for sigmoid modeling which does not assume the same inflection point as do Cq (Ct) based methods by using a fixed threshold to determine Cq (Ct) for all amplifications...
See: http://www.cy0method.org/
Another interesting thing to note here (when the dRn axis is in log scale plotted versus cycle), if you rotate the picture 90 degrees counter clockwise (so that dRn is the x axis and Cycle # is on the y axis, each amplification plot is its own standard curve: the slope of line drawn through the actual inflection point which is indicative of the efficiency of the amplification (a slope of -3.3219 being 100% efficiency in log base 10 vernacular, or -1 in log base 2 vernacular). But, in order for this to be accurate and valid, the correct inflection point has to be chosen/calculated (which is the exact center of the line through which the tangent is drawn): this is something that the Richards equation (from 1959) is still the best at determining. Exactly as used in the Cy0 method. In this method, you are indeed relying on dRn to determine the parameters.
- Badges
- Science topic