NORTHERN BLOTTING

IQRA JABBAR

M.PHIL.

SCHOOL OF BIOLOGICAL SCIENCES

WHAT IS BLOTTING?

Proteins, DNA or RNA, can be transferred onto a carrier

nitrocellulose or nylon membrane

The transfer of biological samples from a gel to a membrane and their subsequent detection on

the surface of the membrane

Blotting Types

Southern(DNA)

Northern(RNA)

Western (Protein)

TYPES OF BLOTTING

NORTHERN BLOTTING

A technique to detect and measure specific

RNA sequences

Developed by James Alwine and George Stark

at Stanford University in 1977

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Oligo (dT) column chromatography can be done to isolate only polyA+ RNAs

Formaldehyde used as denaturing agent in gels to remove secondary structures.

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Nylon membranes are preferred • positively charged• high capacity to bind

nucleic acids• greater robustness on

handling)

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cDNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.

Probes can be Radioactive (Radiolabelled with P32) Or Chemiluminiscent (probe attached to enzyme for chemiluminiscent substrate or to a ligand..)

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Steps involved in northern blotting

BLOTTING SET UP

Transfer buffer contains formamdie• It lowers the annealing temperature for probe-

RNA • Prevents RNA degradation at high

temperatures.

CHEMILUMINISCENT PROBES

APPLICATIONS OF NORTHERN BLOTTING

To observe a particular gene’s expression

pattern between

Tissues Organs Developmen

tal stages

Environmental stresses

Pathogen infection

APPLICATIONS OF NORTHERN BLOTTING

Comparison of

expression of

oncogenes and tumor

suppressor

genes between

cancerous and normal

tissues

Normal Cancerous

Oncogene

Tumor suppressor

gene

APPLICATIONS OF NORTHERN BLOTTING

By using only one probe.. If obtain

variance in the level of each

band….

• May be alternative spliced

products

• Or repetitive sequence motifs

• Deletion or errors in transcript….

Can be tested by changing

probe target sequence… find the

missing regionSingle probe is used for detection

ADVANTAGES

High specificity

Quality and quantity of RNA can be determined

Probes with partial

homology can be used

Membranes can be

stored and re-probed

DIS-ADVANTAGES

Dis-advantages

Small number of genes can be

dealt at one time

Degradation by RNases

Use of harmful chemicalsLow sensitivity

Detection with multiple probes is

difficult

Thank you