Gene expression
DNA (Genome)
pre-mRNA
mRNA
mRNA (Transcriptome)
Proteins (Proteome)
Metabolites(Metabolome)
Regulation
Nucleus
Cytoplasm
• Chromatography• Mass spectrometry• NMR
• DNA arrays and chips• (semi) qRT-PCR• Northern blot + hybrid.• Transkriptional fusions
Functionalgenomics
• 2D electrophoresis Mass spectrometry Protein sequencing• Translational fusional• Immunodetection• Enzyme activities
• Genome maping• Genome sequencing• Genome annotations
Structuralgenomics
Transcriptome
- set of all mRNAs present in certain cell, tissue, organ, …
- mRNA level results from intensity of transcription and mRNA stability
Transcriptomics – expression analysis of populations of genes- analysis of differences in expression of gene populations(under different conditions, treatments, developmental stages)
Analysis of gene transcription – mRNA level
DNA (Genome)
mRNAs(Transcriptome)
Proteins(Proteome)
Microarrays
Transcriptional fusion of gene promotor with reporter gene
Macroarrays
Real time PCRqRT-PCR;Semiquant.RT-PCR
P P gene TT
Hybridization on Northern blots
Reporter gene
Methods based on hybridization
Methods based on PCR
1. Transcriptional fusion of promoter with reporter gene encoding glukuronidase or GFP
- easy analysis of the sites of certain gene expression in planta
Arabidopsis thaliana
P P gen TT
Reporter gene
Compare with promoter-trap mutagenesis !
qRT-PCR a Semiquantitative RT-PCRoriginal level of template measured as:
- PCR product level after certain number of cycles- number of PCR cycles necessary to reach certain product
level
qRT(real time)-PCR
mRNA isolation
Reverze transcription(oligo T-primer, specific reverze
primer)
Semiquantitative RT-PCR
cDNA
Electrophoretic detection limit
Proper number of cycles has
to be determinedfor semiq. RT-PCR
qReal Time - PCRDetection of product level – fluorescent probes improve specificity
R …reporterQ …quencher D … donorA … acceptor
Fluorescent labels:
Principle of detection of nucleic acids by hybridization
Probe - strand of NA with known sequence used for detection
of complementary strand in a mixture of NAs (e.g. transcripts, cDNAs, genomic fragments)
Two phases system (): hybridization of complementary single-stranded NA:
immobilized (bound on membrane, glass) mobile phase (NA in solution)
immobilized probes(on known positions)
mobile, labeled mixture of NA
Arrangement I:
Arrangement II:immobilized mixtures
mobile labeled probe
- labelling by usually by incorporation of labelled nucleotide during NA synthesis
Types of labeling – radioactive (most frequently 32P)
- fluorescent
- digoxygenin, biotin etc. + (followed by detection with a specific antibody)
Labeled probes for hybridization
Hybridization on Northern blots
MicroarraysMacroarrays
RNA isolation
Electrophoretic separation
Blotting = transfer of mRNA from gel onto a membrane
Hybridization with labelled probe, detection
Hybridization on Northern blots
Immobilized phase – analyzed mixture of mRNAs
Mobile phase = labeled probe of certain gene
(signal = presence of certain transcript + info about the transcript size)
x Hybridization on DNA arrays or chips
Immobilized phase – multiple probes with known sequences bound on certain places of the solid support
Mobile phase = labeled mixture of analyzed NAs(simultaneous detection of presence and quantity of many sequences)
Fluorescent (RI) signal
Pozition
Identity
Intensity
Amount
DNA arrays and DNA chips - principle
Array, chip(imobilized probe)
Fluorescently (RI)labelled analyzed NAs(mobile phase)
Hybridization
Terminology: arrays, chips
Macroarray (High DensityArray)
Preparation Support Density[probes/cm2]
Printing of oligo-nucleotids or PCR fragments
Membrane max. 64
Microarraye.g. glass up to 104
Chip Direct synthesis on the support
e.g. glass up to 2.5 *105
Printing of oligo-nucleotids or PCR fragments
ArraysProbes
Imobilized probes
cDNA (ESTs)
Genome sequences
Oligonucleotides, …
Mobile phase (usually labelled cDNA) Imobilized phase (array)
Situation II
Situation I
RNA isolation Labelling
Hybridization
Comparison of gene expression using differential labelling on arrays
Alternative approach: independent hybridization and comparison of the results
1. Non-specific (cross-) hybridizations, background
2. Signal intensity depends also on sequence (differences in efficiency of hybridization)
3. Reproducibility
Troubles with hybridization on arrays
• every probe on different positions on the array• several different probes for every gene
• Affimetrix chips
Solutions:
Oligonucleotide chips from Affymetrix- mutiple probes for every gene (20 pairs), direct synthesis on the chip
- probes from 3' end of mRNA (for Eucaryots)- every oligonucleotide in perfectly matching version and with one missmatch
mRNA sequence
Perfect match
Single NT missmatch
Gene sequencePairs of oligonuceotide probes
Perfect match
Single NT missmatchFluorescens intensity
Differences in fluorescence intensity between perfect and missmatched oligonucleotide are averaged for all probe pairs
5‘ 3‘
Sample labeling for hybridization on Affimetrix chips
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
mRNA
AAAAAAAAATTTTTTTTTT- T7 promoter
cDNA with T7 promoterreverze
transcription
UUUUUUUUUB
B
BB
UUUUUUUUUB
B
BB
UUUUUUUUUB
B
BB
UUUUUUUUUB
B
BB
Biotin-labelled cRNA
in vitro transcription with biotinilated NTP
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
Sample ready for hybridization with the chip
fragmentation
Ligation with promoter
Affymetrix chips - hybridization and result analysis
BBB B
B
B BB
B
BBB B
B
B BB
BB
streptavidin- phycoerythrin binds to biotin
BBB B
B
B BB
BB
detector
excitation at 488 nm
Emission at 570 nm
Image analysis
B
Genevestigatorhttps://www.genevestigator.com
partially free approach to chip results
- species- genes- chips (experiments)
Selection of: