2. Blotting
Process in detecting any
macromolecule that we deal with
it
Macromolecule may be DNA, RNA
or Protein
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3. Blotting
IF we are detecting DNA we called
it Southern Bloting
IF we are detecting RNA we called
it Nothern Bloting
IF we are detecting Protein we
called it Western Boting
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4. Northern Blotting
Technique developed in 1977 or 1979 by
J.Alwine, D. Kemp & G. Start
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5. Northern Blotting
Very Similar to Southern Bloting
Technique based on Nucleic acid
Hybridization
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Nucleic acid hybridization: A technique in which single-
stranded nucleic acids(DNA or RNA) are allowed to interact so
that complexes called hybrids are formed by molecules with
similar, complementary sequences.
6. Northern Blotting (Importance)
To Study gene expression by detecting RNA
in a Sample During differentiation,
morphogenesis as well as abnormal or
diseased condition
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7. Northern Blotting (Importance)
Detects the presence a specific mRNA
in a total RNA extract
Can determine whether a gene is
transcribed or not
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8. Northern Blotting (Stages)
Major stages involve in Northern Bloting
To Prepare target
Gel electrophoresis
Blot
Hybridization
Detection
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9. Northern Blotting (Process)
To Prepare target
Target is RNA
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10. Northern Blotting (Process)
Simply Extract the content of cell that contain
RNA and than Purify RNA Extract from other cell
components
RNA Extraction and purification takes place by
Trizol
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11. Northern Blotting (Process)
After Extraction and Purification
Treat RNA with different endonucleases
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12. Northern Blotting (Process)
Endonulceases are Restriction endonucleases
Cut the RNA into different Fragments
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13. Northern Blotting (Process)
But the cutted Fragments of RNA are present in
Secondary Structure because whenever the find
complementary regions, they bind
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15. Northern Blotting (Process)
For Detection they must be Linear
TO Solve this problem we use DENATURING
GEL ELECTROPHORESIS
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16. Northern Blotting (Process)
DENATURING GEL ELECTROPHORESIS
Agarose gel with extra formaldehyde is used for
this purpose
DENATURING GEL
ELECTROPHORESIS
Separate the fragments on the basis
of size
And linear the RNA
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17. Northern Blotting (Process)
Extra Formaldehyde makes it (RNA Fragments)
linear
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23. Northern Blotting (Process)
After the Separation of fragments on the basis of
size
We will go for Blotting
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24. Northern Blotting (Process)
Blotting
IMPRINTING gel material on to the paper
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25. Northern Blotting (Process)
In Blotting
We Transfer the content of gel onto the paper
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26. Northern Blotting (Process)
In this Blotting
We use Whitman's Filter paper No. 52
Amino Benzoxy Methyl In Place of Nitrocellulose
membrane Filter Paper (Southern Blotting)
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27. Northern Blotting (Process)
Amino Benzoxy Methyl
Better transfer medium for RNA
More binding affinity towards RNA
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28. Northern Blotting (Process)
Transfer the content of gel onto the paper is takes
place by through capillary action
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29. Northern Blotting (Process)
Capillary action transfer the content of gel (RNA)
from gel to the paper (Amino benzoxy methyl)
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32. Northern Blotting (Process)
After transferring the content of gel (RNA) from
gel to the paper (Amino benzoxy methyl)
We Exclude everything out and take membrane
out
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33. Northern Blotting (Process)
Put the membrane into a solution containing
probes
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34. Northern Blotting (Process)
Probe will Hybridize only on a specific Target
mRNA
In case of Southern Bloting DNA-DNA duplex is
formed
In case of Northern Bloting RNA-DNA duplex is
formed Probe
Short sequence of Nucleotide that is
complementary to its target
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35. Northern Blotting (Process)
Bath the membrane with another solution that
would not be containing a probe
BY THIS WAY all probes will be removed except
target hybridized probe
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36. Northern Blotting (Process)
After probe Hybridization
We will go for Detection
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37. Northern Blotting (Process)
IF probe is radioactively labeled, we go for Radio
autography
IF probe is Fluorescent, we go for
chemiluminescence
IF probe is Colori, we go for colorimetric analysis
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38. Northern Blotting (Process)
IF probe is radioactively labeled, we go for Radio
autography
IF probe is Fluorescent, we go for
chemiluminescence
IF probe is Colori, we go for colorimetric analysis
We put X-ray
Band is formed on X-ray
Probe will Emit Light
Probe Will Emit color
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40. Comparison Between
Northern Blotting & Southern Blotting
Southern Blotting Northern Blotting
DNA molecules
detected
Agarose gel electrophoresis
DNA/DNA hybridization
Detecting systems are
Colori, Radioactive,
Chemical
Probe is ssDNA
Blotting method is capillary
action
Nitrocellulose membrane is
RNA molecules detected
Agarose denaturing gel
electrophoresis with extra
formaldehyde
DNA/RNA hybridization
Detecting systems are
Colori, Radioactive,
Chemical
Probe is ssDNA
Blotting method is capillary
action
Amino benzoxy methyl
membrane is used
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42. RNA EXTRACTION BY TRIZOL
(Process)
Add 1ml trizol to the sample and homogenize
Add 2ul chloroform to homogenate
Vortex vigorously
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43. RNA EXTRACTION BY TRIZOL
Incubate on ice for 15 minutes
Centrifuge to get phase separation
12,000g for 15minutes at 4 degree centigrade
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g is the relative
centrifugal force
44. RNA EXTRACTION BY TRIZOL
Transfer the aqueous phase to a fresh tube
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45. RNA EXTRACTION BY TRIZOL
Precipitate the RNA by mixing with 0.5ml
isopropanol
Incubate on ice for 10 minutes
Centrifuge for 10minutes at 12,000g at 4 degree
centigrade
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46. RNA EXTRACTION BY TRIZOL
Remove the supernatant (Surface Liquid)
Wash pellet with 1ml 70% ethanol by flicking
Centrifuge at 7500g for 10minutes at 4 degree
centigrade
Pellet
Supernat
ant
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47. RNA EXTRACTION BY TRIZOL
Remove supernatant
Air dry the Pellet (RNA Pellet)
Dissolve RNA Pellet in appropriate volume of
Rnase-free water
Now further we treat this RNA with restriction
endonucleases
And cutted into Fragments
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