Transcript quantificationcihr2003.ppt

Quantification of fluorescent signals
Sabine Mai, Ph.D.
Manitoba Institute of Cell Biology
Goals:
Reliable analysis of data
Comparison of fluorescent and molecular data
Valid conclusions
Precision of data
Special goals for this workshop:
Rules of quantifications - relationship to other methods
- relate this experience to own research program..
Reliable analysis of data using fluorescence
Qualitative vs.quantitative analysis
FISH signals: how many and where?
how intense (deletion vs. amplification)
Protein signals: where? How many?
How much protein is expressed?
Controls: what are appropriate controls?
Comparison of fluorescent and molecular data
FISH: how does it relate to other techniques? How can it be compared?
Single cell vs. whole cell population.
Precision and combination with other techniques.
Morphology.
Protein: how does it relate to other techniques?
Protein analysis of individual cells and of populations
Protein localization studies.
Precision.
Morphology.
Valid conclusions.
Data can be measured and compared between research and
clinical laboratories.
Data are stored and archived and can be revisited any time.
Specific software makes analysis independent of user:
valid, reliable, objective.
Precision of data.
•Individual genes,
•protein localization and movement,
•chromosome structure and changes,
•three-dimensional (3D) localization within the
interphase nucleus.
Spectral Imaging
Qualitative analysis: genomic rearrangements.
Controls:
SKY on primary cells (normal cells).
Additional methods:
M-FISH, painting, FISH, Southern.
Three-dimensional analysis of the interphase nucleus.
Trisomy 11.
Measurement of relative positions within the nucleus in mm scale.
QuickTime™ and a
Intel Indeo® Video 5.0 decompressor
are needed to see this picture.
Controls:
Pirmary cell with normal chromosome 11.
Additional methods:
None.
Chromosome painting.
Measurement of length of duplicated chromosome bands.
Controls:
Normal cells (primary cells) without chromosomal
changes.
Additional methods:
Southern blot
Centromere FISH.
Evaluation of centromere numbers and sizes.
Controls:
Internal control for centromere length
Additional methods:
Cytometry analysis of centromere length by FACS.
Q-FISH.
Measurement of telomere length.
Controls:
Internal control for telomere length
Additional methods:
Southern blot to measure relative telomere length
Cytometry analysis of telomere length by FACS.
Fluorescent immunohistochemistry.
Measurement of protein levels and
protein location.
Controls:
- Antibody controls
- Cells that serve as positive and negative controls.
Additional methods:
Western analysis.
Fluorescent immunohistochemistry.
Single cell and population analyses.
Controls:
- Antibody controls
- Cells that serve as positive and negative controls.
Additional methods:
Western analysis.
Disadvantages:
- no single cell analysis, only population
analysis;
- simultaneous analysis of several parameters is not
possible;
- localization of protein(s) is not as obvious.Contamination
issues;
- morphology of cell(s) is absent in Western blot.
Goals for the workshop:
Acquisition of images and analysis on different workstations.
Focus today: telomeres, protein, and later 3D analysis.
Analysis/measurements with different software packages:
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Northern Eclipse
Applied Imaging
Teloquant
Please see Kim for the distribution of groups
at different workstations
and computers.