From the technical point of view, you can analyze the methylation status in each animal individually. All you need is a few nanogramms (50 to 200) of bisulfite treated genomic DNA which you can amplify by PCR. Then you can either directly sequence or subclone and sequence about 10 individual clones which if there is no bias will be derived from both alleles with statistical equal chances. This will be of course only important if you expect allele-specific differences.

Otherwise if you expect no differences within your group you can also combine up to your own consideration, pools of animals with different age groups which will will facilitate the procedure of course. But in order to get a statistical chance to analyze all the different alleles in this pool, it will be important to analyze more PCR products (for example number of animal multiplied by ten).

For the latter procedure, you could also treat each genomic DNA of an animal individually with bisulfite and only combine it at the step before doing pcr. So if you  find differences within your pool you can come back to the individual DNA and analyze it later on.

Good luck with your experiments,

Leo

Hi,

This kind of problems do self clear themselves. I will suggest that to have a much better analysis of the work and the methylation status, you should have a regular interval of ages, distributed in at least five nodal points. If the gene that you are looking at is a cell regulatory one, then with age it may change. So an age group would be good. Better, since you are doing methylation analysis and if is from the blood of the animal, then design an experiment such that you house the animal and collect the blood samples at regular intervals, just to nullify the several external aspects like genetic variability in methylation profile determination. Epigenetics is very dynamic and you need atleast 30 samples and experiments to be triplicated to believe your results.

Hope this will help you.

Best,

Somnath.

 From the technical point of view, you can analyze the methylation status in each animal individually. All you need is a few nanogramms (50 to 200) of bisulfite treated genomic DNA which you can amplify by PCR. Then you can either directly sequence or subclone and sequence about 10 individual clones which if there is no bias will be derived from both alleles with statistical equal chances. This will be of course only important if you expect allele-specific differences.

Otherwise if you expect no differences within your group you can also combine up to your own consideration, pools of animals with different age groups which will will facilitate the procedure of course. But in order to get a statistical chance to analyze all the different alleles in this pool, it will be important to analyze more PCR products (for example number of animal multiplied by ten).

For the latter procedure, you could also treat each genomic DNA of an animal individually with bisulfite and only combine it at the step before doing pcr. So if you  find differences within your pool you can come back to the individual DNA and analyze it later on.

Good luck with your experiments,

Leo

You may follow the both advise. For animal study grouping, please consider following points

Equal number of animals in each group.

Strain and sex of animals in the group should be same.

For good results, you should analyse the individual samples....

If your pooling the samples then consider same number for all the groups.. (not 4 and 6)

Hope this will help you...

Sushant

Question 1: I don't know how to organize my groups of animals. In literature, this part is poorly developed so I don't know if I can look at the methylation status of pool(s) of animals or animals individually.

I know that there are very well written publications in this field - look for the ones that are well-written (and be more careful criticizing the publications of people in your field...)! I am quite sure that details are well-described well in the publication I was part of attached below (Kovacheva et al.). If you have any more questions please message me directly here on RG I will be glad to help.

However, as a quick answer for all, usually each animal is measuerd individually, and then the results are combined/pooled afterwards if necessary, as already stated by Leopold Fröhlich. This of course means (and I would seriously recommend) doing the bisulfite treatment individually for each DNA. After this you have the option of pooling for measurment (which I would not recommend, unless inevitable), or measuring and then pooling results.  The latter is the more common and what we do in our lab. There are, of course, exceptions: if material is limited, for example when investigating oocytes, then you may be forced to pool before bisulfite treatment or measurement to have enough material - but this should not be the case for rat tissues, which are comparatively large. 

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Question 2: The first experiment I did was one pool of 6 young rats and one pool of 4 old rats, but I don't know if this is correct, if I have to replicate with other pools or if I have to assess the methylation status of each animal individually.

For a first-off check I guess this would have been ok. It would have been better if you had measured animals individually, as pooling may obscure small differences. The consequence that I think this has for you is this: if you see differences between young and old in your analysis as it is now, it makes sense to do a more strictly planned second analysis. However, if you do not see differences, it may be because pooling is hiding small differences.

In general I would say there are better ways to approach a longitudinal study/ study over time. For example, you could draw blood from the same animals at different ages, as Somnath suggested. That way you can follow the methylation of each animal over time. This will not be possible if you need to sacrifice the animals for your analyses. Then it would be best to compare siblings from the same litter (or at the very least form the same mating pair), and sacrifice some at young and some at old age. You will need setups like these as otherwise other factors, such as the differences between animals of one strain, may influence/obscure your results too heavily and hide the methylation increase over time that you are looking for. 

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Question 3: I think this method is more qualitative than quantitative.

There are qualitative and quantiative ways to measure bisulfite-treated DNA. You do not state which method you are using after bisulfite conversion. I guess it is Sanger sequencing? If this is the case you will need to sequence mulitple clones and quantify based on that - it will not be possible to quanitfy based on the peaks of only electropherogram! Could you please go a bit more into detail? Then I can tell you more :-)

Hope this helps and good luck with your analyses!

Nick

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