NBlot
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maulidyaNBlot
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maulidyaHybridization
n Formation of probe-
probe-target complex, e.g. DNA-
DNA-
DNA, DNA-
DNA-RNA or Protein-
Protein -Protein
n Used to identify target molecules among a
large population of similar molecules
n Three primary gel-
gel-based methods
u Southern blot - DNA
u Northern blot - RNA
u Western blot - protein
Northern Blotting
n Purpose - to identify mRNA that is expressed or
repressed under specific conditions
u control vs
vs.. pollution exposed
u developmental expression
1
No RNases
n RNA easily degraded
n RNases are omnipresent
n extremely resistant
Rules for RNA Work
n ALWAYS USE GLOVES!!!
n ALWAYS USE GLOVES!!!
n ALWAYS USE GLOVES!!!
n NO BARE HANDS!!!
n NO BARE HANDS!!!
n NO BARE HANDS!!
2
Northern Blotting
Overview
n Eight general steps
u 1) RNA isolation
u 2) gel electrophoresis *
u 3) transfer to membrane *
u 4) probe labeling
u 5) blocking
u 6) hybridization
u 7) washing
u 8) detection
RNA Isolation
n Disruption/ Cell Lysis
Lysis// Denaturation
u thiocyanate, $ - Mercapto ethanol
Guanidinium thiocyanate,
n Extraction
u acid phenol, chloroform, CsCl
n Precipitation
u LiCl,, ethanol, isopropanol
LiCl
n Spin columns
u GI lysis + silica-
silica- gel
gel membrane purification
3
RNA Isolation
n Trizol
u solution of phenol and guanidine isothiocyanate
u add chloroform, centrifuge
t separates into aqueous and organic phases. RNA
remains in aqueous phase.
u isopropanol precipitation
Chironomus tentans
n Non-biting midge fly
Non-
n 28 day life cycle
u egg 4 instar stages pupae adult
4
Northern Blotting - Day 1
n Electrophoresis
n Blotting
n Probe labeling
Gel Electrophoresis
n Separates based on size
5 3 A
5
n Denature RNA 3 B
u ensures that separation is based on molecular weight
u prevents H-
H- bonding between base pairs
t Formamide gel
t Glyoxal
Glyoxal/DMSO
/DMSO
t Methyl Hg
t Urea
5
RNA Gel
28s 28s/18s
18s
tRNA
tRNA
From: Qiagen revolutions in RNA purification
C. tentans
Blotting
n Transfer RNA to solid support (membrane)
u nitrocellulose or nylon
u electrophoresis or capillary blotting
paper towels
filter paper direction of transfer
membrane
gel
10X SSC
6
Probes
n DNA
u cDNA isolated by PCR
t directly from PCR sample
t band cut from an agarose gel
n RNA
Probe Labeling
n Radiolabel
n Antigen labeling
u digoxigenin (DIG) coupled to dUTP
t a hapten derived from the steroid digoxin (Digitalis
lanata))
lanata
u random primed labeling
t DNA is heat denatured
t add mixture containing random hexamers
hexamers,, dNTPs
dNTPs,,
DIG-- dUTP
DIG dUTP,, Klenow Enzyme (polymerase
(polymerase fragment)
t incubate overnight at 37o C
7
Random Primed Labeling
Roche Molecular Biochemicals
Day 2
n Immobilize RNA to blot by UV crosslinking
(covalently binds RNA to membrane)
n Prehybridize blocks unbound surfaces of the
membrane
n Hybridize to probe overnight
u buffer contains formamide or urea, SDS, SSC,
and blocking agent
8
Factors Affecting Hybridization
n Complementarity correct base pairs stability
n Base pair types stability linearly with %GC
n Salt concentration
u negative chg. on phosphate backbone must be
neutralized to form duplex
u higher salt (SSC) = stringency
n Temperature
u () G)
affects energy of formation ()
u temperature = less favorable reaction
Day 3
n Wash membrane
u removes unbound and loosely bound probe
u change stringency to allow or prevent mismatches
n Detection
u autoradiography
u immunodetection alkaline phosphatase
9
DIG-Mediated Northern Blot
BCIP NBT
Roche Molecular Biochemicals
Final Product
HSC70
Actin
10
Rules for RNA Work
n ALWAYS USE GLOVES!!!
n ALWAYS USE GLOVES!!!
n ALWAYS USE GLOVES!!!
n NO BARE HANDS!!!
n NO BARE HANDS!!!
n NO BARE HANDS!!
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