Northern blotting

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 1 12/2/2016

2. Blotting Process in detecting any macromolecule that we deal with it Macromolecule may be DNA, RNA or Protein Roll No. 12 (Mujahid Hussain) M.Phil Botany 2 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 3 12/2/2016

4. Northern Blotting Technique developed in 1977 or 1979 by J.Alwine , D. Kemp & G. Start Roll No. 12 (Mujahid Hussain) M.Phil Botany 4 12/2/2016

5. Northern Blotting Very Similar to Southern Bloting Technique based on Nucleic acid Hybridization Roll No. 12 (Mujahid Hussain) M.Phil Botany 5 12/2/2016 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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 6 12/2/2016

7. Northern Blotting (Importance) Detects the presence a specific mRNA in a total RNA extract Can determine whether a gene is transcribed or not Roll No. 12 (Mujahid Hussain) M.Phil Botany 7 12/2/2016

8. Northern Blotting (Stages) Major stages involve in Northern Bloting To Prepare target Gel electrophoresis Blot Hybridization Detection Roll No. 12 (Mujahid Hussain) M.Phil Botany 8 12/2/2016

9. Northern Blotting (Process) To Prepare target Target is RNA Roll No. 12 (Mujahid Hussain) M.Phil Botany 9 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 10 12/2/2016

11. Northern Blotting (Process) After Extraction and Purification Treat RNA with different endonucleases Roll No. 12 (Mujahid Hussain) M.Phil Botany 11 12/2/2016

12. Northern Blotting (Process) Endonulceases are Restriction endonucleases Cut the RNA into different Fragments Roll No. 12 (Mujahid Hussain) M.Phil Botany 12 12/2/2016

13. Northern Blotting (Process) But the cutted Fragments of RNA are present in Secondary Structure because whenever the find complementary regions, they bind Roll No. 12 (Mujahid Hussain) M.Phil Botany 13 12/2/2016

14. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 14 12/2/2016

15. Northern Blotting (Process) For Detection they must be Linear TO Solve this problem we use DENATURING GEL ELECTROPHORESIS Roll No. 12 (Mujahid Hussain) M.Phil Botany 15 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 16 12/2/2016

17. Northern Blotting (Process) Extra Formaldehyde makes it (RNA Fragments) linear Roll No. 12 (Mujahid Hussain) M.Phil Botany 17 12/2/2016

18. Northern Blotting (Process) Smaller Fragments move faster, migrate more, travel more distance Larger Fragments move slower, migrate less, travel less distance Roll No. 12 (Mujahid Hussain) M.Phil Botany 18 12/2/2016

19. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 19 12/2/2016

20. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 20 12/2/2016

21. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 21 12/2/2016

22. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 22 12/2/2016

23. Northern Blotting (Process) After the Separation of fragments on the basis of size We will go for Blotting Roll No. 12 (Mujahid Hussain) M.Phil Botany 23 12/2/2016

24. Northern Blotting (Process) Blotting IMPRINTING gel material on to the paper Roll No. 12 (Mujahid Hussain) M.Phil Botany 24 12/2/2016

25. Northern Blotting (Process) In Blotting We Transfer the content of gel onto the paper Roll No. 12 (Mujahid Hussain) M.Phil Botany 25 12/2/2016

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) Roll No. 12 (Mujahid Hussain) M.Phil Botany 26 12/2/2016

27. Northern Blotting (Process) Amino Benzoxy Methyl Better transfer medium for RNA More binding affinity towards RNA Roll No. 12 (Mujahid Hussain) M.Phil Botany 27 12/2/2016

28. Northern Blotting (Process) Transfer the content of gel onto the paper is takes place by through capillary action Roll No. 12 (Mujahid Hussain) M.Phil Botany 28 12/2/2016

29. Northern Blotting (Process) Capillary action transfer the content of gel (RNA) from gel to the paper (Amino benzoxy methyl) Roll No. 12 (Mujahid Hussain) M.Phil Botany 29 12/2/2016

30. Northern Blotting (Process) Roll No. 12 (Mujahid Hussain) M.Phil Botany 30 12/2/2016

31. Northern Blotting (Process) Amino Benzoxy Me. Weight Amino benzoxy methyl Roll No. 12 (Mujahid Hussain) M.Phil Botany 31 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 32 12/2/2016

33. Northern Blotting (Process) Put the membrane into a solution containing probes Roll No. 12 (Mujahid Hussain) M.Phil Botany 33 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 34 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 35 12/2/2016

36. Northern Blotting (Process) After probe Hybridization We will go for Detection Roll No. 12 (Mujahid Hussain) M.Phil Botany 36 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 37 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 38 12/2/2016

39. Northern Blotting (disadvantages) Roll No. 12 (Mujahid Hussain) M.Phil Botany 39 12/2/2016

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 used 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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 40 12/2/2016

41. 12/2/2016 Roll No. 12 (Mujahid Hussain) M.Phil Botany 41 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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 42 12/2/2016

43. RNA EXTRACTION BY TRIZOL Incubate on ice for 15 minutes Centrifuge to get phase separation 12,000g for 15minutes at 4 degree centigrade Roll No. 12 (Mujahid Hussain) M.Phil Botany 43 12/2/2016   g  is the relative centrifugal force

44. RNA EXTRACTION BY TRIZOL Transfer the aqueous phase to a fresh tube Roll No. 12 (Mujahid Hussain) M.Phil Botany 44 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 45 12/2/2016

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 Supernatant Roll No. 12 (Mujahid Hussain) M.Phil Botany 46 12/2/2016

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 Roll No. 12 (Mujahid Hussain) M.Phil Botany 47 12/2/2016

48. Summary (NORTHERN BLOTTING) 12/2/2016 Roll No. 12 (Mujahid Hussain) M.Phil Botany 48 Prepared target ( Fragmented RNA ) Gel electrophoresis Blotting Hybridization Detection

49. Thank You  Roll No. 12 (Mujahid Hussain) M.Phil Botany 49 12/2/2016