- - - - - - - - - - - - - - Original Message - - - - - - - - - - - - - - From: "Thomas Delohery" <t-delohery@ski.mskcc.org> Subject: Re: Data Analysis Software Date: 03/20/99 18:52 I must be missing something here. I don't understand the point(s) being debated or the issues being raised. But I'll throw my two cents in anyway. Several years ago I was evaluating cell cycle analysis programs; ie, curve-fitting algorithms for analyzing histograms from cells with fluorescent stained DNA. I used BrdU incorporation as my Gold standard. After analyzing numerous data files there was only one program that consistently gave values for percentage of cells in G1, S and G2/M that agreed with the BrdU data. The program always gave a slightly LOWER S-phase (and correspondingly, slightly higher G1 and G2/M) than the BrdU incorporation data. In looking at DNA histograms of BrdU positive cells I noticed they usually showed a fairly discreet G2/M peak. It then occurred to me that the curve-fitting program was giving a BETTER measure of the percentages cell cycle compartments than the BrdU data. BrdU incorporation measures all cells in S-phase over the period of the BrdU pulse. That includes the "dim" BrdU positives, with "apparently G1 DNA content" that are in the earliest stages of S-phase to cells that completed S-phase very soon after the BrdU was added. These would be BrdU positive G2 cells. So the fact that the curve-fitting program was giving values for percent S-phase below those measured by BrdU incorporation makes perfect sense. DNA staining alone can't be resolve late-G1 cells from early-S and late-S from G2. That's why a GOOD curve-fitting algorithm will have an area of overlap at these transitions. It's a little alarming to hear Adam Treister say: >But for me, the role of data analysis software is to enable exploration >and to test >potential models, not only to churn existing models. Mark's message lists >valid uses >for the capability. Clearly there are limitations, but this is flow, so >what else is >new? The software is there only to calculate the numbers; it's up to the >biologist >to make sense of them. One would hope that the software calculations are providing realistic numbers so biologists can make sense of them. Esp. given the fact that I just ordered 3 copies of FlowJo. The last thing I need is a repeat of something like CellFit where the numbers calculated are heavily dependent on the model one chooses to fit the data. I wouldn't recommend you be too quick to discard the old models while you're exploring and testing "potential " models. td -- ============================================================================== Thomas M. Delohery |Internet: t-delohery@ski.mskcc.org Supervisor, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 1275 York Ave. Box 98 | New York, NY 10021 | ============================================================================== Hi, yes I agree with Thomas totally! The BrdUrd technique is really the ONLY standard if it comes to precision. Years ago I was able to measure about 10 sec of DNA sysnthesis time with the monoclonal technique, which is about 1/1000 of the DNA content of a typical cell with 6 hours S-phase duration. there is no technique in the world besides BrdUrd (or other analogs of course), which can recognize that. however we have to pay a price, that's the longer laboratory work we have to do for it. The difference between BrdUrd and other DNA staining with intercalated or whatsoever dyes is, that the signal to be measured starts from zero for BrdUrd and starts with about mor than 10 to the nine molecules in standard DNA staining work. Best regards from Munich Wolfgang Beisker GSF- National Research Center for Environment and Health Ingolstaedter Landstr. 1 85764 Neuherberg Germany
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:17 EST