The document details the northern blotting technique, which is used to detect RNA molecules through nucleic acid hybridization. It outlines the process stages, including RNA extraction, gel electrophoresis, blotting, hybridization, and detection methods. Additionally, it compares northern blotting to southern blotting, highlighting different materials and specific uses for each technique.

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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