Hello
Today i want to get help on how to form graph bar based on qpcr results.
I already know how to calculate fold change.
For example,
Suppose my gene of interest is A and control gene is B
and average cq results are like below :
Control gene A : 27
Control gene B : 15
Experimental gene A : 29.5
Experimental gene B : 15.5
I can calculate fold change like this : (27-15) - (29.5-15.5) = -2
2^-2 = 0.25.
So i can indicate that gene A is downregulated(1/4) after experimental treatment.
But i still cannot figure out how significance of difference(ex) *, **) and standard deviation is structured to complete bar graph of qpcr.
Thank you
Dear Lee,
For your question i suggest to you links and attached files in topics.
-BioTechniques - qPCR: Technical Replicate Variation
www.biotechniques.com › In the Journal › 2011 › January
-Statistical significance of quantitative PCR | BMC Bioinformatics | Full ...
bmcbioinformatics.biomedcentral.com/articles/.../1471-2105-8-13...
-Data Analysis - PCR Technologies Guide | Sigma-Aldrich
www.sigmaaldrich.com/technical-documents/.../data-analysis.html
Best regards
Dear Lee,
If you want to calculate standard deviation and figure out significance of difference, you need to repeat your qPCR experiment with independent biological samples (at least 3 times). You can then compare the fold change you get for each independent experiment, calculate mean, sd and test significance of difference using an appropriate statistical test.
For example, exp1 FC=0.25, exp2 FC=0.35, exp3 FC=0.20, the mean FC is 0.26667, and the sd=0.076. You can use a t-test to check for statistical difference.
You might find this interesting: http://www.nature.com/nature/journal/v492/n7428/full/492180a.html. Talks about when you can/cannot/should/shouldnt calculate error bars, and how to calculate them.
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