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❥NORTHERN BLOTTING


NORTHERN BLOTTING – 30 MARK DETAILED NOTES

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Northern blotting is a molecular biology technique used to detect specific RNA molecules in a complex mixture. It provides information about gene expression, RNA size, and transcript abundance by hybridizing RNA with a labeled complementary DNA or RNA probe.
πŸ“Œ Named by analogy to Southern blotting (DNA detection).


2. Principle


The principle of Northern blotting is based on:
Separation of RNA molecules by size using denaturing agarose gel electrophoresis
Transfer (blotting) of separated RNA onto a nylon or nitrocellulose membrane
Hybridization of membrane-bound RNA with a labeled complementary probe
Detection of RNA–probe hybrids by autoradiography or chemiluminescence
✔ Only RNA sequences complementary to the probe will be detected.


3. Types of RNA Analyzed


mRNA (most common)
rRNA
tRNA
miRNA and siRNA (with modified protocols)

4. Requirements / Materials

Total RNA or poly(A)+ RNA
Denaturing agarose gel (formaldehyde or glyoxal)
Electrophoresis buffer (MOPS)
Nylon or nitrocellulose membrane
Labeled probe (radioactive or non-radioactive)
Hybridization buffer
Washing solutions
Detection system

5. Steps Involved in Northern Blotting


Step 1: Isolation of RNA
RNA extracted from cells/tissues using TRIzol or phenol-chloroform method
RNase-free conditions are essential
Quality checked using agarose gel or spectrophotometer
πŸ“Œ RNA is unstable → RNase contamination must be avoided

Step 2: Denaturing Gel Electrophoresis


RNA mixed with formaldehyde to prevent secondary structures
Loaded onto agarose gel
Separation occurs based on molecular size
✔ Denaturing conditions ensure accurate size separation

Step 3: Transfer of RNA to Membrane (Blotting)
RNA transferred from gel to membrane by:

Capillary transfer
Vacuum blotting
Electroblotting
RNA fixed to membrane by:
UV cross-linking
Baking at 80°C

Step 4: Pre-Hybridization


Membrane incubated in pre-hybridization buffer
Blocks non-specific binding sites
Reduces background noise


Step 5: Hybridization


Membrane incubated with labeled probe
Probe binds to complementary RNA sequence
Conditions: temperature, salt concentration, time
Types of probes:
Radioactive (³²P)
Non-radioactive (biotin, digoxigenin, fluorescent dyes)

Step 6: Washing


Excess and non-specifically bound probe removed
Stringency adjusted using salt concentration and temperature
Step 7: Detection
Radioactive probes → autoradiography (X-ray film)
Non-radioactive probes → chemiluminescence or fluorescence
✔ Bands appear at positions corresponding to RNA size

6. Controls Used


Housekeeping genes (Ξ²-actin, GAPDH)
RNA ladder (size marker)
Negative control probe


7. Interpretation of Results


Band position → size of RNA transcript
Band intensity → level of gene expression
Multiple bands → alternative splicing or multiple transcripts

8. Advantages
Detects specific RNA molecules
Provides information on:
Transcript size
Expression level
Highly specific
Useful in gene expression studies
9. Limitations
Requires large quantity of RNA
Time-consuming
Use of radioactive probes poses safety issues
Less sensitive compared to RT-PCR
RNA degradation affects results
10. Applications
Study of gene expression
Detection of mRNA levels
Analysis of alternative consumption/splicing
Verification of transcriptional regulation
Validation of microarray and RNA-seq data
Diagnosis of viral infections


13. Precautions

Use RNase-free glassware and reagents
Wear gloves
Avoid repeated freeze–thaw of RNA
Maintain denaturing conditions

14. Conclusion
Northern blotting is a classical and reliable technique for studying RNA expression. Though newer techniques like RT-PCR and RNA-seq are more sensitive, Northern blotting remains important for confirming transcript size and integrity.



1. Northern blotting is mainly used to detect
A. DNA
B. RNA
C. Protein
D. Lipids
✅ Answer: B
2. Northern blotting is analogous to which technique?
A. Western blotting
B. Southern blotting
C. Eastern blotting
D. ELISA
✅ Answer: B
3. The principle of Northern blotting is based on
A. Antigen–antibody interaction
B. DNA replication
C. Nucleic acid hybridization
D. Protein folding
✅ Answer: C
4. Which RNA is most commonly detected using Northern blotting?
A. tRNA
B. rRNA
C. mRNA
D. siRNA
✅ Answer: C
5. Which gel is used in Northern blotting?
A. Polyacrylamide gel
B. SDS-PAGE
C. Agarose gel
D. Native gel
✅ Answer: C
6. Why are denaturing agents used in Northern blotting?
A. To increase RNA size
B. To prevent RNA degradation
C. To remove secondary structure
D. To stain RNA
✅ Answer: C
7. Common denaturing agent used in Northern blotting is
A. SDS
B. Urea
C. Formaldehyde
D. Ethanol
✅ Answer: C
8. The membrane commonly used in Northern blotting is
A. Cellulose membrane
B. Nitrocellulose membrane
C. PVDF membrane
D. Glass fiber
✅ Answer: B
9. Which membrane is most preferred today for Northern blotting?
A. Cellulose
B. Nitrocellulose
C. Nylon
D. Agar
✅ Answer: C
10. RNA is transferred from gel to membrane by
A. PCR
B. Capillary transfer
C. Centrifugation
D. Sonication
✅ Answer: B
11. Fixation of RNA on membrane is done by
A. SDS
B. Heat or UV
C. Ethanol
D. Enzymes
✅ Answer: B
12. Which buffer is commonly used in RNA electrophoresis?
A. TAE
B. TBE
C. MOPS
D. PBS
✅ Answer: C
13. The labeled probe used in Northern blotting is
A. Protein
B. Lipid
C. DNA or RNA
D. Carbohydrate
✅ Answer: C
14. Most sensitive radioactive label used is
A. ³H
B. ¹⁴C
C. ³²P
D. ¹²⁵I
✅ Answer: C
15. Hybridization occurs between
A. DNA–DNA
B. RNA–RNA
C. DNA–RNA
D. Protein–RNA
✅ Answer: C
16. Pre-hybridization step is done to
A. Destroy RNA
B. Increase background
C. Block non-specific sites
D. Label RNA
✅ Answer: C
17. Which of the following is a non-radioactive label?
A. ³²P
B. Biotin
C. ¹⁴C
D. ³H
✅ Answer: B
18. Detection of radioactive probes is done by
A. ELISA
B. Autoradiography
C. Western blot
D. PCR
✅ Answer: B
19. Detection of non-radioactive probes is done by
A. Chemiluminescence
B. Centrifugation
C. Electrophoresis
D. Dialysis
✅ Answer: A
20. Band intensity in Northern blot indicates
A. RNA size
B. RNA purity
C. RNA quantity
D. RNA charge
✅ Answer: C
21. Band position in Northern blot indicates
A. Expression level
B. Molecular weight
C. Size of RNA
D. RNA stability
✅ Answer: C
22. Multiple bands in Northern blot suggest
A. RNA degradation
B. Alternative splicing
C. Protein contamination
D. DNA replication
✅ Answer: B
23. Which gene is used as internal control?
A. lacZ
B. pBR322
C. Ξ²-actin
D. Taq polymerase
✅ Answer: C
24. Which blotting technique detects proteins?
A. Northern
B. Southern
C. Western
D. Eastern
✅ Answer: C
25. Which blotting technique detects DNA?
A. Northern
B. Southern
C. Western
D. Eastern
✅ Answer: B
26. Major disadvantage of Northern blotting is
A. Low specificity
B. RNA instability
C. Low accuracy
D. No detection
✅ Answer: B
27. Northern blotting is useful in studying
A. Protein structure
B. Gene expression
C. DNA replication
D. Translation
✅ Answer: B
28. Which enzyme contamination must be avoided?
A. DNase
B. RNase
C. Protease
D. Ligase
✅ Answer: B
29. Which RNA is used as size marker?
A. DNA ladder
B. Protein marker
C. RNA ladder
D. Lipid marker
✅ Answer: C
30. The technique is less sensitive than
A. Southern blot
B. Western blot
C. RT-PCR
D. ELISA
✅ Answer: C
31. Which of the following is NOT a step in Northern blotting?
A. Electrophoresis
B. Blotting
C. Hybridization
D. Translation
✅ Answer: D
32. RNA integrity is checked by
A. SDS-PAGE
B. Agarose gel
C. ELISA
D. PCR
✅ Answer: B
33. Which RNA is least suitable for Northern blotting?
A. mRNA
B. rRNA
C. tRNA
D. DNA
✅ Answer: D
34. High stringency washing results in
A. More binding
B. Less specificity
C. Removal of mismatched probes
D. RNA degradation
✅ Answer: C
35. Transfer by electric field is called
A. Capillary blotting
B. Vacuum blotting
C. Electroblotting
D. Dot blotting
✅ Answer: C
36. RNA is negatively charged due to
A. Sugar
B. Nitrogen base
C. Phosphate group
D. Hydrogen bonds
✅ Answer: C
37. Housekeeping genes are used to
A. Detect RNA
B. Normalize expression
C. Label probes
D. Degrade RNA
✅ Answer: B
38. Northern blotting was developed by
A. Edwin Southern
B. James Watson
C. Alwine et al.
D. Frederick Sanger
✅ Answer: C
39. Which molecule prevents RNA secondary structure?
A. NaCl
B. Formaldehyde
C. EDTA
D. Tris
✅ Answer: B
40. The major role of hybridization buffer is
A. RNA separation
B. Probe binding
C. RNA labeling
D. Membrane fixation
✅ Answer: B
41. Which blot is used for post-translational modification analysis?
A. Northern
B. Southern
C. Western
D. Eastern
✅ Answer: D
42. Northern blotting is a
A. Immunological technique
B. Biochemical technique
C. Molecular biology technique
D. Microbiological technique
✅ Answer: C
43. The transfer membrane must be
A. Hydrophobic
B. Positively charged
C. Negatively charged
D. Neutral
✅ Answer: B
44. Which RNA tail is present in eukaryotic mRNA?
A. Phosphate tail
B. Poly(A) tail
C. Poly(U) tail
D. Poly(G) tail
✅ Answer: B
45. Northern blotting cannot detect
A. RNA size
B. RNA expression
C. Protein activity
D. Transcript variants
✅ Answer: C
46. Use of radioactive probe is discouraged because
A. Low sensitivity
B. High cost
C. Health hazards
D. Poor binding
✅ Answer: C
47. Slot blot is a modification of
A. Western blot
B. Southern blot
C. Northern blot
D. Eastern blot
✅ Answer: C
48. Which step reduces background signal?
A. Electrophoresis
B. Pre-hybridization
C. RNA isolation
D. Transfer
✅ Answer: B
49. Which molecule is complementary to mRNA probe?
A. DNA
B. Protein
C. Lipid
D. Carbohydrate
✅ Answer: A
50. Northern blotting is best described as
A. Protein detection method
B. RNA expression analysis technique
C. DNA sequencing method
D. Enzyme assay
✅ Answer: B


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