Northern blotting
Probe labeling
pBS-SemaIII
dATP dTTP dGTP
RNA isolation
dCTPAAAAAA
Gel electophoresis
Blotting
Hybridization
Autoradiography
reverse Northern blotting
Down-regulation of transcripts Up-regulation of transcripts
* *
labeled (specific) probe target
Northern
* *
specific probes
labeled target
reverse Northern
cDNA arrays (continued)
• macro-arrays
– low-density
– filter-supported – radioactive probes
(duplicates) – low sensitivity
– only cDNA clones spotted
– cheap
cDNA arrays (continued)
• micro-arrays
– high-density – glass-supported – fluorescent probes
(single slide) – high sensitivity – ability to spot
oligonucleotides
– very expensive
Tessuto della prostata,
normale Tessuto della prostata,
tumorale
Un microarray è un supporto solido sul quale sono stati posizionati diverse migliaia di cDNA in spot separati. Ciascuno spot rappresenta un gene, in quanto contiene numerose copie di un cDNA corrispondente a tale gene.
I microarrays
In questo esempio i cDNA
sono stati posizionati su di un
vetrino, simile ai normali
vetrini usati per l’istologia.
Microarray technology
http://www.ym.edu.tw/excellence/HBP/HBP_CP4/procedure.htm
Microarray synthetized by photolithography or EST/oligo “linking”
Probes
si confrontano i profili di espressione genica di due campioni differenti.
E’ necessario estrarre le molecole di mRNA dai due campioni.
Tessuto della prostata,
normale Tessuto della prostata,
tumorale
utilizzo dei microarrays
Arrays Probe DNAs are “spotted”
onto a glass slide
Each spot corresponds to a particular gene e.g. β-actin
Each array will have
thousands of spots (typically
3000 to 30000)
Targets RNA is extracted from tissues or cells
test reference
mRNA
cDNA RNA is copied to DNA
labelled DNA is fluorescently labelled
pooled Samples are pooled
Hybridisation
Labelled target DNA hybridised to array
Fluorescence of each spot indicates how much of
particular RNA was present in
both samples
Data Processing
Hybridised Hybridised
Array Array
Images Images
Scanner
Ratios Ratios
Spot-finding
Normalisation
Normalised Normalised
Ratios Ratios
Further
analysis
Cy5 Cy5 Cy3
Cy3 Cy3
test reference Cy5
