BIO198L Gene Biotechnology Laboratory

1st Quarter SY 2015-2016

Northern Blot
Lomugdang, Fiord Jogardy B.1

1Student (s), Subject/Section, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology

ABSTRACT

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.In this module the paper of separating RNA by gel electrophoresis and transferring
it to a filter is very similar to that used for DNA, We use northern blotting to compare the RNA from normal individuals with the RNA from
patients with the diseases under investigation by choosing RNAs for the northern blot, selecting a fragment of DNA to use as a probe,
hybridize the northern blot with the probe and interpret the hybridization pattern.

INTRODUCTION

The northern blot is a technique used in molecular biology

research to study gene expression by detection of RNA (or I. Choose RNAs to fill the lanes in the gel
isolated mRNA) in a sample. With northern blotting it is possible to
observe cellular control over structure and function by determining 1. Choose RNAs for each lane of the Southern blot. The first lane
the particular gene expression rates during differentiation and of the gel contains gel marker RNA.
morphogenesis, as well as in abnormal or diseased conditions 2. Continue to select RNAs that are relevant to the disease
Northern blotting can be used to analyzed a sample of RNA from investigating
a particular tissue or cell type in order to measure the RNA
expression of particular genes. II. Cross link RNA to the filter

-globin is a component (subunit) of a larger protein called 1. Use the forceps to peel away the gel by dragging it from raised
hemoglobin, which is located inside red blood cells. The HBB gene corner. Drag it to trash can.
provides instructions for making a protein called -globin. 2. To fix the RNA to the nylon filter, place it under UV light in the
Hemoglobin normally consists of four protein subunits: two UV crosslinking oven
subunits of -globin and two subunits of another protein called 3. Drag the filter to the oven.
alpha-globin. Each of the four protein subunits of hemoglobin 4. Set time of UV exposure for 10 seconds
carries an iron-containing molecule called heme. Heme molecules 5. Turn on the oven by clicking ON/OFF switch
are necessary for red blood cells to pick up oxygen in the lungs
and deliver it to cells throughout the body. A complete hemoglobin III. Set up the prehybridization
protein is capable of carrying four oxygen molecules at a time
Oxygen attached to hemoglobin gives blood its bright red color. 1. Place the filter into the roller bottle after UV exposure.
2. Transfer 10 mL hybridization solution from flask to roller bottle
Northern blotting was developed by James Alwine and George 3. Drag the roller bottle to the hybridization oven.
Stark at Stanford University and was named such by analogy to 4. Set temperature to 47 degrees Celsius.
Southern blotting. Northern blotting involves the following steps: 1) 5. Set time to 1 hour
Total cellular RNA or mRNA is size-separated by denaturing
agarose gel electrophoresis, 2) The separated RNA is transferred
onto a nylon membrane; 3) The RNA is then detected with isotopic
or non-isotopic labelled complementary DNA or RNA probe. The
objective of this experiment is to make the reader understand or
know the basic principles of northern blotting and its significance. IV. Select a radioactively labeled probe
MATERIALS AND METHODS

Experiment 07 Date: September 18 2015 1 of 3

 BIO198L Gene Biotechnology Laboratory
1st Quarter SY 2015-2016
1. Select radioactively labeled probe.
V. Denature the probe
1. Drag the probe tube to the heat block.
2. Set temperature to 98 degrees Celsius
3. Set incubation time for 5 minutes
VI. Add the probe to the roller bottle
1. Transfer 25 microliters of the radioactively labeled probe to the
roller bottle with filter.
2. Drag the capped roller bottle into hybridization oven.
3. Set temperature to 42 degrees Celsius.
4. Set time to 4 hours.
VII. Wash the filter
1. Pour off the hybridization solution into the radioactive waste
bottle.
2. Transfer 10 milliliters of hybridization wash solution from beaker
to the roller bottle.
3. Place roller bottle into hybridization oven.
4. Set temperature to 60 degrees Celsius
5. Set time to 1 hour.
VIII. Place the filter in the X-ray cassette
1. Drag the filter to the X-ray cassette.
2. Click the top of the X-ray cassette.
3. A filter will expose the X-ray film
4. Interpret the results.
RESULTS & DISCUSSION
Experiment 07 Date: September 18 2015
A general blotting procedure starts with extraction of total RNA
from a homogenized tissue sample or from cells. Eukaryotic
mRNA can then be isolated through the use of oligo (dT) cellulose
chromatography to isolate only those RNAs with a poly(A) tail RNA
samples are then separated by gel electrophoresis. Since the gels
are fragile and the probes are unable to enter the matrix, the RNA
samples, now separated by size, are transferred to a nylon
membrane through a capillary or vacuum blotting system.
The RNA samples are most commonly separated on agarose gels
containing formaldehyde as a denaturing agent for the RNA to limit
secondary structure. The gels can be stained with ethidium
bromide and viewed under UV light to observe the quality and
quantity of RNA before blotting.
Northern blotting is a means of identifying a certain RNA species
in a complex mixture of RNA. Commonly used to evaluate
qualitatively and quantitatively the expression of a gene, Northern
blotting involves isolation of RNA, size fractionation of the
denatured RNA through gel electrophoresis, transfer of the
separated RNA to a membrane, hybridization with a specific
probe, and detection.
The first major consideration is that RNA is both chemically and
biologically far more labile than DNA. From a practical viewpoint
the extreme sensitivity of RNA to RNases and the wide prevalence
and stability of these enzymes means that in order to prepare
intact molecules of RNA, one must pay careful attention to
eliminating RNases. Secondly, only a few percent of the total RNA
of a cell is m-RNA, so that blotting against total RNA is not a very
sensitive way of detecting rare mRNA. Therefore, although one
can often detect a target mRNA in a preparation of total RNA, one
can attain greater sensitivity by isolating first the poly A+ mRNA
fraction and carrying out Northern analysis. Ten mg of total RNA
is sufficient to detect an abundant message, whereas several mg
or more of poly A+ RNA is generally required to detect rare
messages. Poly A+ RNA can be isolated directly or from total RNA
by means of oligo dT cellulose run either in a column or batch
mode, by magnetic beads crosslinked to oligo dT, or by strepavidin
2 of 3
 BIO198L Gene Biotechnology Laboratory
1st Quarter SY 2015-2016

coated magnetic beads capable of binding biotinylated oligo which CONCLUSION

has been hybridized to mRNA.
A northern blot is a laboratory method used to detect specific RNA
molecules among a mixture of RNA. Northern blotting can be used
to analyze a sample of RNA from a particular tissue or cell type in
order to measure the RNA expression of particular genes. This
method was named for its similarity to the technique known as a
Southern blot. -thalassemia is a genetic disorder wherein the -
globin protein is not primarily being produced or is being reduced
because of improper mRNA sequencing. Patients with
dysfunctional -globin genes may suffer from sickle cell anemia
and -thalassemia. Northern blotting is not very effective when it
comes to detection of genetic diseases but it is effective in
detecting certain mutations in the genetic makeup particularly the
mRNA. -globing probe was used to determine the -globin RNA
present in each of the patients. The objectives of the experiment
were met.

REFERENCES

Moffatt, B (2006). Course Notes Biology 208.Waterloo, University

Figure 1. UV Image
of Waterloo.

Dale, J, & von Schantz, M (2003). From Genes to

Genomes.West Sussex: John Wiley & Sons Ltd.

GDL Gene Discovery Lab

Figure 2. X-ray image

Experiment 07 Date: September 18 2015 3 of 3