Transcript Transkriptom a proteom - Univerzita Karlova v Praze

Transcriptome
and analysis of gene transcription
Gene expression
• Genome maping
• Genome sequencing
• Genome annotations
Structural
genomics
Nucleus
DNA (Genome)
pre-mRNA
Cytoplasm
• DNA arrays and chips
• (semi) qRT-PCR
• Northern blot + hybrid.
• Transkriptional fusions
mRNA
mRNA (Transcriptome)
Proteins (Proteome)
Metabolites
(Metabolome)
• 2D electrophoresis
Mass spectrometry
Protein sequencing
• Translational fusional
• Immunodetection
• Enzyme activities
• Chromatography
• Mass spectrometry
• NMR
Functional
genomics
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
Methods based
on hybridization
Macroarrays
Hybridization on
Northern blots
Microarrays
Methods based on PCR
mRNAs
(Transcriptome)
Real time PCR
qRT-PCR;
Semiquant.
RT-PCR
DNA (Genome)
Proteins
(Proteome)
Reporter gene
P
gene
T
Transcriptional fusion of gene
promotor with reporter gene
1. Transcriptional fusion of promoter with
reporter gene encoding glukuronidase or
GFP
Reporter gene
P
gen
T
- easy analysis of the sites of certain gene expression in planta
Arabidopsis
thaliana
Compare with promoter-trap mutagenesis !
qRT-PCR a Semiquantitative RT-PCR
original level of template measured as:
- PCR product level after certain number of cycles
- number of PCR cycles necessary to reach certain product level
mRNA isolation
Semiquantitative
RT-PCR
Reverze transcription
(oligo T-primer, specific reverze
primer)
cDNA
qRT(real time)-PCR
Proper number
of cycles has
to be determined
for semiq. RT-PCR
Electrophoretic detection limit
qReal Time - PCR
Detection of product level – fluorescent probes improve specificity
Fluorescent labels: R …reporter
Q …quencher
D … donor
A … acceptor
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
Arrangement I:
(on known positions)
mobile, labeled mixture of NA
immobilized mixtures
Arrangement II:
mobile labeled probe
Labeled probes for hybridization
- 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)
Hybridization on Northern blots
RNA isolation
Electrophoretic separation
Macroarrays
Microarrays
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)
DNA arrays and DNA chips - principle
Hybridization
Fluorescent (RI)
signal
Fluorescently (RI)
labelled analyzed NAs
(mobile phase)
Pozition
Array, chip
(imobilized probe)
Identity
Intensity
Amount
Terminology: arrays, chips
Preparation
Support
Macroarray
(High Density
Array)
Printing of oligonucleotids or
PCR fragments
Membrane
e.g. glass
Microarray
Printing of oligonucleotids or
PCR fragments
Direct synthesis on
the support
e.g. glass
Chip
Density
[probes/cm2]
max. 64
up to 10
4
up to 2.5 *10
5
Arrays
Probes
Mobile phase
(usually labelled cDNA)
Imobilized phase (array)
cDNA (ESTs)
Imobilized probes
Genome sequences
Oligonucleotides, …
Automated preparation of macroarrays contact printing
4.5 mm
Comparison of gene expression
using differential labelling on arrays
Situation I
RNA isolation
Hybridization
Labelling
Situation II
Alternative approach:
independent hybridization and comparison of the results
Identification of differentially expressed genes
Troubles with hybridization on arrays
1. Non-specific (cross-) hybridizations, background
2. Signal intensity depends also on sequence
(differences in efficiency of hybridization)
3. Reproducibility
Solutions: • every probe on different positions on the array
• several different probes for every gene
• Affimetrix chips
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
3‘
5‘
Pairs of oligonuceotide probes
Gene sequence
Perfect match
Single NT missmatch
Perfect match
Fluorescens intensity
Single NT missmatch
Differences in fluorescence intensity between perfect and missmatched
oligonucleotide are averaged for all probe pairs
Sample labeling for hybridization on Affimetrix chips
reverze
transcription
mRNA
AAAAAAAAA
cDNA with T7 promoter
AAAAAAAAA
TTTTTTTTTT- T7 promoter
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
Ligation with promoter
in vitro transcription with biotinilated NTP
B B
B
B
B B
B
B
B
B
B
B
B
B
fragmentation
B
B
B
Sample ready for hybridization with the chip
B
UUUUUUUUU
B B
UUUUUUUUU
B
B B
UUUUUUUUU
B
B
B
B
B
B B
B
B
B
Biotin-labelled cRNA
B
B
UUUUUUUUU
Affymetrix chips - hybridization and result analysis
BB B
B
B
B
B
B B
B
streptavidin- phycoerythrin binds
to biotin
BB B
B
B
B
B
B B
B
Image analysis
detector
Emission at 570
nm
BB B
B
B
B
B
B B
excitation
at 488 nm
B
Genevestigator
https://www.genevestigator.com
partially free approach to chip results
Selection of:
- species
- genes
- chips
(experiments)
Affymetrix chip preparation
Photolithography