This document describes the process of northern blotting. Northern blotting is used to detect specific RNA molecules and analyze gene expression. It involves separating RNA samples by size using gel electrophoresis, transferring them to a membrane, then using a labeled probe to detect the RNA of interest. The key steps are isolating RNA from tissue or cells, separating the RNA by size on a gel, blotting the RNA onto a membrane, hybridizing the membrane with a labeled probe, washing off excess probe, and visualizing where the probe bound to detect the RNA of interest. Northern blotting allows researchers to study gene expression patterns in different tissues, developmental stages, or disease states.

1. BY: NUSRAT SHEIKH

2.  INTRODUCTION
 STEPS IN NORTHERN BLOTTING
 PROCEDURE
 APPLICATIONS
 DISADVANTAGES

3. WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like
DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting

4. NORTHERN BLOTTING
 A northern blotting is a laboratory method used to detect specific RNA
molecules among a mixture of RNA (mRNA).
 The technique was developed in 1979 by James Alwine and his colleagues.
 Northern blotting can be used to analyze a sample of RNA from a particular
tissue or cell type in order to measure the expression of particular genes.
 Northern blotting involves the use of electrophoresis to separate RNA samples
by size, and detection with a hybridization probe complementary to part of or
the entire target sequence.
 The term ‘northern blot’ actually refers specifically to the capillary transfer of
RNA from the electrophoresis gel to the blotting membrane. However the
entire process is commonly referred to as northern blotting.

5. RNA isolation
Separation of RNA’s using
gel electrophoresis
Blotting
Hybridization with
Labelled probe
Washing off excess probes
Visualization

7. 1. RNA isolation:
The blotting procedure starts with isolation of total RNA from a homogenized
tissue sample or from cells. Eukaryotic mRNA can be isolated through the use of
oligo (dT) cellulose chromatography to isolate only those RNAs with poly (A) tail.
2. Separation of RNA using gel electrophoresis:
RNA samples are then separated by agarose gel electrophoresis containing
formaldehyde as a denaturing agent for RNA to limit secondary structure. An
electric current is passed through the gel and the RNA moves away from the
negative electrode. The distance moved depends on the size of the RNA fragment.
Since genes are of different Sizes, the size of RNA varies. This results in a smear
on the gel.

8. 3. BLOTTING:
RNA is single stranded, so it can be
transferred out of the gel and onto a filter
membrane. Aminobenzoxymethyl filter
membrane is used for northern blotting
as it has more binding affinity towards RNA.
The RNA molecules retain the same pattern
of separation.
4. Hybridization with labelled probe:
It is the process of forming a ds DNA-RNA hybrid molecule
between a ssDNA probe and a ss target RNA.
There are 2 important features of hybridization:
i. The reactions are specific i.e. the probes will only bind to targets wih a
complementary sequence.
ii. The probe can find one molecule of target in a mixture of millions of related
but non-complementary molecules.
Commonly cDNA is created with labelled primers for the RNA
sequence of interest to act as the probe in the northern blot.

9. The probes must be labelled either with radioactive isotope or with
chemiluminescence. X-ray film can detect both the radioactive and
chemiluminescent signals. Thus, following hybridization, the probe permits the
RNA molecule of interest to be detected from among the many different RNA
molecules on the membrane.
5. WASHING OFF EXCESS PROBES:
The probe is bound specifically to the target RNA and that there is negligible
non-specific binding to other RNA. The excess probes in the membrane is
washed off.
6. VISUALIZATION:
RNA-DNA hybrid can be visualized and detected by autoradiograph. A band
should be observed on the autoradiograph if the probe has hybridized to a
stretch of RNA on the filter.

10.  Northern blot is a valuable method used by researchers in determining gene
expression patterns between tissues, organs, developmental stages, pathogen
infection and over the course of treatment.
 The technique has been used to show overexpression of oncogenes and down
regulation of tumor-suppressor genes in cancerous cells when compared to normal
tissue.
 The variance in size of a gene product can indicate deletions or errors in transcript
processing.
 By altering the probe target used along the known sequence it is possible to
determine which region of the RNA is missing.
 Used in the screening of recombinants by detecting the mRNA produced by the
transgene.

11.  In northern blotting only one or a small number of genes can be visualized at a
time.
 Specific cell cannot be studied.
 Main problem in northern blotting is often sample degradation by Rnases,
which can be avoided by proper sterilization of glasswares and the use of Rnase
inhibitors such as DEPC.
 The chemicals used in most northern blotting can be a risk to the researcher,
since formaldehyde, radioactive material, ETBR, DEPC and UV light are all
harmful under certain exposures.

12. THANK-YOU