My understanding is that other species than your study-species can be used. Is it better to get a sample of the study-species as an internal standard, or will any sample of known chromosome-number do? Trying to learn about how the flow cytometry method works, and what I need to use it. My study-species are invasive Fallopia spp. 4X, 6X and 8X individuals have previously been found in Europe.
Hi, Anne-Kari,
Do you mean DNA contents in pg per geniome?
Plesae, remebre that you have to stain your materail with excatly same dye concentration as stardad.
you can find detailed answer in this paper: http://www.ncbi.nlm.nih.gov/pubmed/10330063
Good luck!
Hi, Anne-Kari,
Do you mean DNA contents in pg per geniome?
Plesae, remebre that you have to stain your materail with excatly same dye concentration as stardad.
you can find detailed answer in this paper: http://www.ncbi.nlm.nih.gov/pubmed/10330063
Good luck!
Hi, I think a sample of the study-species as internal standard will serve you the best. Thanks
Hi, Anne-Kari, do not forget alos one importnat point: you keep constant all you scanning parameters all of the time. In this case X-axis give you relative value of DNA contents. To get absolute value you need made a calibration with 4-5 known nucleus ea. with know DNA contents in pg..
But it is crucial either made calibration for each run, or keep all parameters constant. Good luck!
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Thank you very much both of you, for good tips and link to a very interesting and relevant paper!
Even I am very much interested on plant genome size estimation. Is this technique helpful to distinguish between two species?
Hello Biswajit : )
Genome size can be used to distinguish between species if 1) the intraspecific variation in genome size is low, and 2) there is sufficient divergence among different species. Flow cytometry is also useful to identify hybrids and their origin, because of their intermediate fluorescens intensity compared to parental species.
Suda et al (2010) Genome size as a marker for identifying the invasive alien taxa in Fallopia section Reynoutria (http://www.preslia.cz/P101Suda.pdf)
Hello Biswajit,
yes, flow cytometry can be useful for distinguish between species,
however, one should consider also that fluorescence intensity does not shown DNA contents, but DNA stain-ability, ea. proportional amount of the dye which able to binding to nucleus. In the case of some species DNA modification (chromatin condensation level etc) were different in different organs even in the same plants, so, you should be very carefully with data interpretation: you should use similar organs with similar DNA stain-ability, if you want to separate 2 species.. Good luck!
Thank you Dr. Pasternak and Anne-Kari Holm for very valuable suggestions... I will keep that in mind.
Hi I am working on a similar topic but on plants In order to determine the ploidy you must use a sample of the same species whose chromosome number/ploidy is known as an external standard (standard run separately) or as an internal standard (sample and external standard run simultaneously) The DNA-selective fluorochrome used also determines the success of the analysis. DAPI (AT) rich dye is usually used but not suitable for determination of nuclear DNA content. I refer you to the work of .Dolezel and Bartos, 2005,and Dolezel et al 1998
Hi, Omena, do not frogert alos to use the same chromatin stage: DNA staiabilty is daramatically dependent from cells type and stages. Good luck!
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