❥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.

📌 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

📌 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 (β-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. β-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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