My experimental protocol is as follows:
I have two groups:
- CTL group (sleep and wake ad libitum), rats euthanized in six different ZTs --> ZT0, ZT4, ZT8, ZT12, ZT16 e ZT20.
Sleep Deprivation group (SD) --> Same Zts as described above.
I performed gene expression by real time PCR using the 2- deltaCt method. Then, I analyzed the data using one way-ANOVA for each group.
When there was statistical significance, I analyzed using COSINOR (El Temps software) as below:
Ex:
CTL
ZT0 group mean
ZT4 group mean
ZT8 group mean
ZT12 group mean
ZT16 group mean
ZT20 group mean
SD
ZT0 group mean
ZT4 group mean
ZT8 group mean
ZT12 group mean
ZT16 group mean
ZT20 group mean
Is that correct? Is there a way to include standard deviations in this analysis?
Dear Ana Dias
Find the answer in the following article
Article Sexual Dimorphism in Circadian Physiology Is Altered in LXRα...
Regards
Dear Ana,
You have 2 groups CTL and SD. You have taken the 2-ddct value for the both groups of each samples. From this valus you can calculate the mean, standard deviation and standard error mean. Then you have applied one-way ANOVA for showing statistically significant or not. You calculate the mean and SD value separately in each sample and each groups.
For the conisor analysis you need the mean and SEM values of each samples of each groups.
Cosinor analysis was used by best curve fitting of the gene expression data to check rhythmicity of the gene across different time points using the following formula:
f(t) =A+ (B x Cos [2 x pi x(t-C)/24]
Where f(t) = gene expression level at a given time, A = mesor (rhythm adjusted mean), B = amplitude (half of the difference between minimum and maximum levels of transcripts), t = time in hours, and C = acrophase
I am using Graphpad prism in this software you have to fit this equation. Then using this formula you will getting the all values, such as mesor, amplitude and acrophase in your experimental groups.
Then you prepare cosinor table including mesor, amplitude, acrophase and p value.
I hope this will help you.
Best Wishes,
Dear Ana Dias
Find the answer in the following article
Article Sexual Dimorphism in Circadian Physiology Is Altered in LXRα...
Regards
I agree with Saurav Saha, but you need to use ANOVA ONE WAY, POST TEST TUCKEY. Theestandar desviation of acrophase, mesor and amplitud you need to analize each value individual for mesor acrophase and amplitude and MAKE a STUDENT TEST.
Previous discussion in Questions and Answers:
https://www.researchgate.net/post/Does_anyone_know_a_method_of_cosinor_analysis#view=544558d7d685cc893a8b45ab
My "popular answer" gave the tunning for graphpad prism, 4 years ago.
Another program to use is the GraphPad Prism 5 with "XY analisys", "Nonlinear Regression (curve fit)". Using this Equation: Y=Baseline+Amplitude*cos(Frecuency*X+Phaseshift). Rules for initial values Baseline: 1 RULE: *YMID Amplitude: 0.5 RULE: *(YMAX-YMIN) Frequency: 0.2618 RULE: (Initial value, to be fit) Phaseshift: 1.0 RULE: *(Valueof X at YMAX) Defaults contrainsts Frecuency Constrains equal to: 0.2618 Baseline, Frecuency and Phaseshft : NO CONSTRAINTS Any questions? [email protected] You obtain the next results. "Best-fit values" " Baseline" " Amplitude" " Frecuency" " Phaseshift" "Std. Error" " Baseline" " Amplitude" " Phaseshift" "95% Confidence Intervals" " Baseline" " Amplitude" " Phaseshift" "Goodness of Fit" " Degrees of Freedom" " R square" " Absolute Sum of Squares" " Sy.x" Constraints " Frecuency" "Number of points" " Analyzed".
I will send you an email explaing in detail.
I use I use cosinor to calculate amplitud, acrophase and mesor. For circadian rhythms I use a software from this page.
This web is not working but I can send you by email.
http://www.chronopharmacology.de/software.htm
"Chronos Fit" software gIves you if you have or not a circadian rhythms, if it is ultradian or circadian, and gives you % of rhythm.
Then using the cosinor software you should analize each value individualy and then make a student analisys using graphpad prism. It will give you the standart desviations. Make sure you change acrophase from 360º to 24 hs, by making rule of 3. 15 degress equivalent to and hour, and the others degress that not fit in and hour you may multiplicate for 60 and divide by 15 (degrades). And the answer gives minutes.
I will send you an email with software you should use graphpad 5 because is more easy to use. Graphpad 6 and 7 need more specific tunning for make ANOVA ONE WAY post TEST TUCKEY.
Respect to your research in Vitamin B deficiency I send you paper from our laboratory researching VAD, Vitamin A Deficiency.
Article Daily rhythms of catalase and glutathione peroxidase express...
Article Circadian rhythms of locomotor activity and hippocampal cloc...
I give you my personal email [email protected] or [email protected]
I will send you an Research Gate message with all the information in detail.
how to analysez circadien heart rate variability using Cosinor logiciel?
Nicolás Andrés Deyurka, I tried the instructions you have for Prism. Very helpful. However, the acrophase value calculated does not correspond to the observed peak time on the nonlinear fit curve. Do you know why this is happening? Thanks!
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