Northern blotting (RNA Dectection by botting process) Southern and northern blotting comparison (similarities and differences)

1. Assignment
Recombinant DNA
Technology
Topic: Northern Blotting
Submitted to: Dr. Ameer Khan
Submitted by: Mujahid Hussain
Roll Number: 12
Class: M.Phil
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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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14. Northern Blotting (Process)
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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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18. Northern Blotting (Process)
 Smaller Fragments move faster, migrate more,
travel more distance
 Larger Fragments move slower, migrate less,
travel less distance
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19. Northern Blotting (Process)
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20. Northern Blotting (Process)
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21. Northern Blotting (Process)
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22. Northern Blotting (Process)
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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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30. Northern Blotting (Process)
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31. Northern Blotting (Process)
Amino Benzoxy
Me.
Weight
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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39. Northern Blotting (disadvantages)
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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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41. 12/2/2016Roll No. 12 (Mujahid Hussain) M.Phil Botany41
RNA EXTRACTION
BY TRIZOL

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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48. Summary (NORTHERN BLOTTING)
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 Prepared target ( Fragmented RNA)
 Gel electrophoresis
 Blotting
 Hybridization
 Detection

49.  Thank You 
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