Transcriptomeand analysis of gene transcription

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)

Automated preparation of macroarrays - contact printing

4.5 mm

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

Identification of differentially expressed genes

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:

Affymetrix chip preparationPhotolithography