Many thanks to Nigel Blackhall, Gerhard Nebe von Caron, Doug Redelman and Dennis J. Young for answering my questions about genome size assessment. Here a short resume of their wisdom: Summary of answers: It is apparently not unusual to periodically see a shift in staining intensity of PI stained samples. To control for this always include a reference in the sample (e.g. chicken RBC nuclei) or better analyse the samples together in the same tube, if possible. Use high concentrations of PI for minimal effect of concentration disturbances. (20 - 50 ug/ml) To dilute samples always use the staining buffer. Optimal flow rates conditions: 500 bugs / sec. It is very important that all samples have the exactly same treatement. In particular cross-linking agents such as formaldehyde reduce the labeling by intercalating dyes such as PI. Be aware that PI binds to chitin and other things in the cell wall, which can alter signal intensity. Original question: Dear flowers, I was asked to determine the genome size of Phytophtora porri, by measuring the emission of its propidium iodide (PI) stained DNA and comparing it to the emission of P.Infestans and E.Coli, for which the genome size is known. My problem now is, that I do not trust the results for following reasons: - The Phytophtora and the E. Coli were not treated exactly the same way. Phytophtora was formaldehyde fixed prior to 70% ethanol treatment while E. Coli was treated with 70% EtOH. only. Both were treated with DNAse-free RNAse and stained with 20ug/ml PI, though. Might the difference in treatment lead to wrong results? - When we measure cell cycle (e.g. in human and mouse cell lines), for reasons that are beyond my understanding, now and then some samples which were treated exactly the same way as the other samples will show a shift of the G0/1 and G2/M peaks compared to the other samples. (The cells are guaranteed to have the same amount of DNA. And I saw this with different users and protocols.) The results are interpretable, but we have to change the location of the markers (which sometimes is quite difficult, if you have a shift of the G1 peak and you do not see exactly where the G2/M peak is). This led me to the conclusion that PI staining might not be ideal to assess the size of the genome, because of inconsistency of staining. - When measuring the E.Coli-PI emission I had to dilute them (with BD FACS Flow) because there were too many of them in the sample. The diluted bacteria showed a shift to the left of almost one log in emission. Now either the FACScan will overestimate the fluorescence when charged with too many cells or, more probably, the staining with PI is very sensitive to dilutions. So the inconsistencies in the cell cycle explained above could be due to a drop of sheet fluid falling into the sample. Again many thanks for the answers Claudio Vallan =================================================== Claudio Vallan PhD Phone Lab: 031 / 632 88 76 FACS-LAB DKF Phone Office: 031 / 632 99 68 University of Bern E-Mail: vallan@dkf7.unibe.ch c/o Institute of Pathology Murtenstrasse 31 Insel hosptial area only: 3010 Bern Beeper: 181 67 59 ===================================================
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