ABSTRACT
Possibility of nonspecific hybridizations • ++
➚ ➙ ➫
Reduction in nonspecific hybridizations • +++
➘ ➘ ➫
Possibility of denaturation 9. Time
• <3 h
➘ ➘ ➫
Incomplete hybridization • 3 h
➚ ➫
Sufficient hybridization • >3 h
➙ ➚ ➫
Risk of an increase in nonspecific hybrids
Criteria
1. NaCl concentration • +++
➚ ➫
Stability of nonspecific hybrids • +
➚ ➘ ➫
Denaturation of nonspecific hybrids 2. Temperature
• +
➚ ➚ ➫
Stability of hybrids • +++
➘ ➘ ➫
Denaturation of hybrids 3. Time
➘ ➘ ➫
Labilization of hybrids
Criteria
1. Autoradiographic
➚ ➚ ➫
Sensitivity 2. Immunohistological
➘ ➘ ➫
Detection threshold • Direct method
➘ ➚ ➫
Nonspecific adsorption • Indirect method
➚ ➙ ➫
Increase in sensitivity • Amplification
➚➚ ➚ ➫
Lowering of the detection threshold
Criteria
1. Bright field
➙ ➙
2. Dark field
➚ ➚ ➫
Amplification of the signal 3. Fluorescence
➘ ➘ ➫
Presence of a threshold 4. Epipolarization
➚ ➘ ➫
Attenuation of a diffuse signal 5. Confocal microscope
➚ ➫
Lowering of the detection threshold 6. Image analysis
➚➚ ➘➘ ➫
Signal analysis, quantification
Criteria
1. Type • cDNA
➘ ➙ ➫
Rehybridization of complementary strands • Single-stranded DNA
➙ ➙ ➫
Hybridization of probe with its target • Oligonucleotide
➙ ➙ ➫
Hybridization of probe with its target • Oligonucleotides
➚ ➙ ➫
Increase in potential hybrids • cRNA
➚ ➙ ➫
High specific activity, stability of hybrids
2. Homology • 100%
➙ ➙ ➫
Optimal conditions • <100%
➘ ➘ ➫
Risk of nonspecific hybrids
3. Label • Radioactive
➚ ➙ ➫
No detection threshold • Antigenic
➘ ➘ ➫
Detection threshold, endogenous antigen
4. Specific activity
➫
To check probe labeling • +
➘ ➙ ➫
Detection threshold • ++
➙ ➙ ➫
Saturating concentration • +++
➚ ➘ ➫
Increase in nonspecific signal
5. Purification
➚ ➚ ➫
Decrease in nonspecific reactions
Criteria
1. Condition for sampling
➚ ➚ ➫
Limitation of contamination 2. Preservation of nucleic
acids
➚ ➚ ➫
Limitation of destruction
Criteria
1. Fixation • +
➚ ➘ ➫
Preservation, but potential diffusion, of targets • +++
➘ ➫
Blockage of nucleic acid-protein complexes 2. Deproteinization
➚ ➫
Accessibility of target nucleic acids 3. Acetylation
➘ ➫
Blockage of NH
functions 4. Prehybridization
➚ ➫
Blockage of nonspecific sites 5. Denaturation
• –
➫
Indispensable with DNA targets • + ➙ ➙ ➫ Accessibility of the target
6. Storage • Without H2O ➙ ➙ ➫ Conservation of targets • Without precaution ➘➘ ➫ Loss of targets
Criteria
1. Probe concentration • + ➘ ➚ ➫ Not all targets hybridized • ++ ➚ ➫ All targets hybridized • +++ ➘ ➫ Increase in nonspecific bonds
2. NaCl concentration ≈ 300 mM
➚ ➫ Stabilization of nucleic acid-nucleic acid hybrids
3. Dextran sulfate concentration
➚ ➫ Probe concentration
4. tRNA, DNA concentrations ➚ ➫ Saturation of nonspecific sites 5. Detergents ➚ ➫ Denaturation of nonspecific sites 6. DTT ➚ ➚ ➫ Protection of the label 7. Formamide concentration ➚ ➚ ➫ Facilitation of hybridization 8. Temperature
• + ➚ ➘ ➫ Facilitation of hybridizations • ++ ➙ ➙ ➫ Optimal hybridization • +++ ➘ ➚ ➫ Denaturation of hybrids
9. Time • <3 h ➘ ➚ ➫ Hybridization of some specific hybrids • 3 h ➙ ➚ ➫ Hybridization of all the specific hybrids • >3 h ➚ ➘ ➫ Hybridization beyond the specific hybrids
Criteria
1. NaCl concentration • +++ ➙ ➚ ➫ Stability of nucleic acid-nucleic acid hybrids • + ➘ ➘ ➫ Instability of hybrids
2. Temperature • + ➚ ➘ ➫ Weak denaturation of nonspecific hybrids • +++ ➘ ➚ ➫ Denaturation of nonspecific more than spe-
cific hybrids 3. Time ➘ ➘ ➫ Hydrolysis of hybrids
Criteria
1. Autoradiographic ➚ ➘ ➫ Absence of a detection threshold, autoradiographic background
2. Immunohistological ➘ ➚ ➫ Existence of a detection threshold 3. Methods
• Direct ➘ ➘ ➫ High detection threshold, background • Indirect ➚ ➚ ➫ Lower threshold • Amplification ➚ ➘ ➫ Risk of background
Criteria
1. Bright field ➙ ➙ ➫ Reference 2. Dark field ➚ ➘ ➫ Excellent visualization of the autoradio-
graphic signal 3. Fluorescence ➘ ➙ ➫ Detection threshold 4. Epipolarization ➚ ➚ ➫ Attenuation of background 5. Confocal microscope ➚ ➫ Spatial visualization of the signal 6. Image analysis ➚➚ ➙ ➫ Signal analysis, quantification
1. Search for homologous sequences in a gene bank
➫ Risk of the existence of sequences with strong homology, which may cause the formation of hybrids
2. Hybridization with a “sense” probe
➫ For oligonucleotides and RNA probes
3. Hybridization with a heterologous probe
➫ For all types of probes
4. Probe competition ➫ Between the labeled probe and a large quantity of the same, but nonlabeled, probe: negative hybridization
5. Nondenaturation of a cDNA probe
➫ Verification of the reactivity of the tissue being studied
1. Radioactive ➫ Determination of specific activity 2. Antigenic ➫ Dot blot on membrane
1. Positive tissue ➫ Tissue or cells known to express the target nucleic acid: positive hybridization
2. Negative tissue ➫ Tissue or cells known not to express the target nucleic acid: negative hybridization
3. Destruction of the target by enzymatic treatment: • DNase ➫ Degradation: hybridization < 0 • RNase ➫ Fragmentation of RNA targets (hybridiza-
tion < 0), which must be followed by numerous washings
Criteria
1. Hybridization with a heterologous probe of the same type
➫ Verification of the reactivity of the tissue being studied
2. Hybridization in the absence of a probe
3. Action on stringency ➫ Variation of the NaCl concentration, or of the hybridization temperature and/or washing: modification of the signal
1. Autoradiographic ➫ Determination of background 2. Immunohistological
• Inhibition ➫ Search for endogenous activity: • Endogenous biotin • Endogenous peroxidase • Endogenous alkaline phosphatase
1. Experimental variations in the expression of the target nucleic acid
➫ Determination of background
2. Quantification of the signal
1. Dot blot, Southern blot, Northern blot
➫ Demonstration of the presence of the target nucleic acid in tissue
2. PCR ➫ Determination of the presence or absence of the target nucleic acid
3. In situ PCR ➫ Amplification of the signal 4. Immunocytology ➫ Revelation of the corresponding protein,
starting with the target nucleic acid
Criteria Signal Background Sensitivity Specificity Probes
1. Type • cDNA ➙ ➙ ➘ ➙
• Singlestranded DNA
➚ ➙ ➚ ➙
• Oligonucleotides
x oligo ➙ ➙
• cRNA ➚ ➘ ➚ ➙ 2. Homology • 100% ➙ ➙ ➙ ➙
• <100% ➘ ➚ ➘ ➘ 3. Label • Radioactive ➚ ➚ ➚ ➙
• Antigenic ➘ ➘ ➘ ➘ 4. Specific activity • + ➘ ➘ ➘ ➙
• ++ ➙ ➙ ➙ ➙
• +++ ➚ ➚ ➚ ➘ 5. Purification ➚ ➘ ➚ ➚
Sampling 1. Sampling conditions ➚ ➚ ➚
2. Preservation of nucleic acids ➚ ➚ ➚ Pretreatments
1. Fixation • + ➚ ➘ ➚ ➘
• +++ ➘ ➚ ➘ 2. Deproteinization ➚ ➘ ➚
3. Acetylation ➘ ➘ ➘
4. Prehybridization ➘ ➚
5. Denaturation • 0 0 ➚ 0
• + ➙ ➙ ➙ 6. Storage • Without H2O ➙ ➙
• Without precaution
➘ ➘➘
Hybridization
1. Probe concentration • + ➘ ➘ ➘ ➚
• ++ ➚ ➚
• +++ ➚ ➘ 2. NaCl concentration • ≈ 300 mM ➚ ➚ ➚ 3. Dextran sulfate concentration ➚ ➚ 4. Concentrations of tRNA, DNA, etc. ➘ ➚ 5. Detergents, etc. ➘ ➚ 6. DTT ➘ ➚ ➚ 7. Formamide concentration ➚ ➘ ➚ ➚ 8. Temperature • + ➚ ➚ ➘
• ++ ➚ ➙ ➙
• +++ ➘ ➘ ➚ 9. Time • <3 h ➘ ➘ ➘ ➚
• 3 h ➚ ➙ ➚ • >3 h ➙ ➚ ➚ ➘
Washing 1. NaCl concentration • +++ ➚ ➙ ➚
• + ➚ ➘ ➘ ➘ 2. Temperature • + ➚ ➚ ➚ ➘
• +++ ➘ ➘ ➘ ➚ 3. Time ➘ ➘ ➘ ➘
Revelation 1. Autoradiography ➚ ➚ ➚ ➘ 2. Immunohistology ➘ ➘ ➘ ➚
• Direct method ➘ ➚ ➘ ➘ • Indirect method
➚ ➙ ➚ ➚
• Amplification ➚➚ ➚ ➚ ➘ Observation
1. Bright field ➙ ➙ ➙ ➙
2. Dark field ➚ ➚ ➚ ➘
3. Fluorescence ➘ ➘ ➘ ➙
4. Epipolarization ➚ ➘ ➚ ➚
5. Confocal microscopy ➚ ➚
6. Image analysis ➚➚ ➘➘ ➚➚ ➙
