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Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.

Kim IH, Nagel J, Otten S, Knerr B, Eils R, Rohr K, Dietzel S - BMC Biotechnol. (2007)

Bottom Line: 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.Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus

Comparison of GFP, immunostaining and FISH signals without RNAse digestion, allowing for detection of RNA from the transgene arrays by the FISH probe. For panel labeling see Figure 2. Projections of deconvolved images are shown. Note the larger volume of FISH signals compared to other signals. In this experiment, due to the large differences in signal appearance, pre- and post-FISH signals could not be subjected to automated image processing. Therefore, for this experiment only, post-FISH images were matched to the orientation of pre-FISH images manually.
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Figure 5: Comparison of GFP, immunostaining and FISH signals without RNAse digestion, allowing for detection of RNA from the transgene arrays by the FISH probe. For panel labeling see Figure 2. Projections of deconvolved images are shown. Note the larger volume of FISH signals compared to other signals. In this experiment, due to the large differences in signal appearance, pre- and post-FISH signals could not be subjected to automated image processing. Therefore, for this experiment only, post-FISH images were matched to the orientation of pre-FISH images manually.

Mentions: To estimate the potential contribution of DNA-RNA hybridization to FISH signals, we repeated the four signal comparison but now without RNase digestion. The transgene array includes β-galactosidase reporter genes. X-gal staining revealed expression in about 40 percent of cells. DNA of the complete plasmid used for generation of the transgenic cell line was labeled and applied as FISH probe, to allow for potential DNA-RNA hybridization. Visual inspection revealed that compared to GFP or immunostaining signals, FISH signals were often larger, labeling the volume surrounding the other signals (Figure 5). The anti-GFP immunostaining signal after FISH did not match this increase, resembling the anti-GFP signal before FISH. This argues for a contribution of RNA bound probe to the FISH signal in this experiment without RNase digestion. Since our quantitative image analysis approach was designed for very similar signals, we could not reasonably apply it to this data set.


Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.

Kim IH, Nagel J, Otten S, Knerr B, Eils R, Rohr K, Dietzel S - BMC Biotechnol. (2007)

Comparison of GFP, immunostaining and FISH signals without RNAse digestion, allowing for detection of RNA from the transgene arrays by the FISH probe. For panel labeling see Figure 2. Projections of deconvolved images are shown. Note the larger volume of FISH signals compared to other signals. In this experiment, due to the large differences in signal appearance, pre- and post-FISH signals could not be subjected to automated image processing. Therefore, for this experiment only, post-FISH images were matched to the orientation of pre-FISH images manually.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254608&req=5

Figure 5: Comparison of GFP, immunostaining and FISH signals without RNAse digestion, allowing for detection of RNA from the transgene arrays by the FISH probe. For panel labeling see Figure 2. Projections of deconvolved images are shown. Note the larger volume of FISH signals compared to other signals. In this experiment, due to the large differences in signal appearance, pre- and post-FISH signals could not be subjected to automated image processing. Therefore, for this experiment only, post-FISH images were matched to the orientation of pre-FISH images manually.
Mentions: To estimate the potential contribution of DNA-RNA hybridization to FISH signals, we repeated the four signal comparison but now without RNase digestion. The transgene array includes β-galactosidase reporter genes. X-gal staining revealed expression in about 40 percent of cells. DNA of the complete plasmid used for generation of the transgenic cell line was labeled and applied as FISH probe, to allow for potential DNA-RNA hybridization. Visual inspection revealed that compared to GFP or immunostaining signals, FISH signals were often larger, labeling the volume surrounding the other signals (Figure 5). The anti-GFP immunostaining signal after FISH did not match this increase, resembling the anti-GFP signal before FISH. This argues for a contribution of RNA bound probe to the FISH signal in this experiment without RNase digestion. Since our quantitative image analysis approach was designed for very similar signals, we could not reasonably apply it to this data set.

Bottom Line: 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.Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus