Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.
BACKGROUND
GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however.


RESULTS
We provide a quantitative comparison of all three approaches. We make use of a cell line that contains a transgene array of lac operator repeats which are detected by GFP-lac repressor fusion proteins. Thus we can detect the same structure in individual cells by GFP fluorescence, by antibodies against GFP and by FISH with a probe against the transgene array. Anti-GFP antibody detection was repeated after FISH. Our results show that while all four signals obtained from a transgene array generally showed qualitative and quantitative similarity, they also differed in details.


CONCLUSIONS
Each of the tested methods revealed particular strengths and weaknesses, which should be considered when interpreting respective experimental results. Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.
Kim
Il-Han
IH
University of Heidelberg, BIOQUANT, IPMB, and German Cancer Research Center (DKFZ), Dept. Bioinformatics and FunctionalGenomics, Biomedical Computer Vision Group, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany. i.kim@dkfz-heidelberg.de
Nagel
Jens
J
Otten
Simone
S
Knerr
Boris
B
Eils
Roland
R
Rohr
Karl
K
Dietzel
Steffen
S
D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D004247:DNA