Northern blotting technique
Summary
TLDRThis video tutorial from Shos Biology explores Northern blotting, a technique for detecting specific RNA sequences within a cell. It emphasizes the similarity between Northern and Southern blotting, explaining that the former focuses on RNA while the latter targets DNA. The process involves RNA extraction, denaturing to linearize the molecules, separation using agarose gel electrophoresis, and transfer onto a membrane for hybridization with a complementary DNA probe. The technique is crucial for identifying specific RNA fragments, and the video promises further insights into related blotting methods.
Takeaways
- 📚 Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA.
- 🔍 It is similar to Southern blotting, which detects DNA, and both involve hybridization techniques.
- 🕵️♂️ The name 'Northern blotting' was derived from 'Southern blotting' and does not relate to geographical directions.
- 🧬 The process involves three main stages: extraction of RNA, separation using denaturing gel electrophoresis, and hybridization with a complementary DNA probe.
- 🧪 RNA is extracted from the cell and then broken down into smaller fragments to eliminate complex secondary structures.
- 🔬 Denaturing gel electrophoresis is used to resolve the secondary structure of RNA into a linear form for accurate separation based on length.
- 🧴 Formaldehyde is added to the gel to help resolve the secondary structure of RNA into a linear form for proper separation.
- 📝 Northern blotting uses a different type of membrane for transferring RNA from the gel compared to Southern blotting, often using aminooxy methy filter paper for better RNA binding.
- 💧 The transfer of RNA from the gel to the membrane is facilitated by capillary action, with the setup including a buffer, sponge, gel, membrane, and paper towels.
- 🔦 A radioactive or chemiluminescent probe is used to bind to the specific target RNA, allowing for the detection and identification of the RNA of interest.
- 📉 The final step involves developing an autoradiograph or using a detection method that reveals the location of the target RNA within the membrane.
Q & A
What is the main purpose of Northern blotting?
-Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA extracted from cells.
What is the relationship between Southern blotting and Northern blotting?
-Southern blotting and Northern blotting are similar techniques, with the main difference being that Southern blotting is used for detecting DNA, while Northern blotting is used for detecting RNA.
Who discovered the Southern blotting technique?
-The Southern blotting technique was discovered by Edwin Southern in 1975.
What is the significance of using formaldehyde in the gel during Northern blotting?
-Formaldehyde is used in the gel to help resolve the secondary structure of RNA into a linear form, which is necessary for accurate separation and detection of RNA fragments.
Why is aminooxy methy paper preferred over nitrocellulose paper in Northern blotting?
-Aminooxy methy paper has a greater binding affinity towards RNA compared to nitrocellulose paper, making it a better medium for transferring and detecting RNA during Northern blotting.
What is the role of the probe in Northern blotting?
-The probe in Northern blotting is a complementary DNA strand that binds specifically to the target RNA sequence. It is often labeled with a radioactive or chemiluminescent molecule to facilitate the detection of the target RNA.
How does the denaturing gel electrophoresis process help in Northern blotting?
-Denaturing gel electrophoresis helps to break down the complex secondary structures of RNA into linear forms, allowing for the separation of RNA fragments based on their length.
What is the purpose of the buffer in the blotting setup?
-The buffer in the blotting setup facilitates the capillary action that is necessary for transferring the RNA from the gel to the membrane or filter paper.
Why is the RNA transferred from the gel to a membrane or filter paper?
-The RNA is transferred to a membrane or filter paper because agarose gel is fragile and not suitable for the probing process, which requires the application of various solutions and buffers.
What is the final step in detecting the target RNA after the transfer to the membrane?
-The final step involves adding the probe to the membrane, which binds specifically to the target RNA. The detection is then achieved through the signal given by the radioactive or chemiluminescent label on the probe.
Why is DNA typically used as a probe in Northern blotting instead of RNA?
-DNA is typically used as a probe in Northern blotting because it has better interaction with RNA and is more stable than RNA, which can easily degrade. The DNA probe is designed based on the complementary RNA structure.
Outlines
🧬 Introduction to Northern Blotting
This paragraph introduces the concept of Northern blotting, a technique used to detect specific RNA sequences within a mixture. It emphasizes the importance of understanding Southern blotting as a basis for Northern blotting due to their similarities. The speaker recommends watching a previous video on Southern blotting for those unfamiliar with blotting techniques. Northern blotting was developed to detect RNA, in contrast to Southern blotting, which detects DNA. The process involves three main stages: preparing the target RNA, using hybridization to find a specific sequence, and employing a probe that can bind to the target RNA. The speaker explains the use of complementary DNA strands as probes, which can be labeled with radioactive or chemical markers to identify the target RNA's position in a gel after separation.
🔬 Denaturing Gel Electrophoresis in Northern Blotting
The second paragraph delves into the specifics of breaking down the secondary structures of RNA into a linear form, which is essential for nucleic acid probing and hybridization. The speaker describes the use of denaturing gel electrophoresis to resolve these structures and separate the RNA fragments based on their length using agarose gel electrophoresis. The paragraph also explains the importance of using formaldehyde in the gel to linearize the RNA and the process of transferring the separated RNA from the gel to a more robust medium, such as aminooxy methy paper, which has a higher binding affinity for RNA than nitrocellulose paper. This transfer process, known as blotting, is critical for the subsequent probing steps.
🌱 Capillary Action in Blotting Techniques
This paragraph explains the process of transferring RNA from the agarose gel to the membrane filter using capillary action, a technique common to both Northern and Southern blotting. The speaker details the blotting setup, which includes a buffer, sponge, gel, membrane, and paper towels, and describes how the buffer flows through the layers due to the applied weight. The RNA fragments are pushed from the gel onto the membrane, where they adhere due to the filter paper's impermeability to RNA. The buffer continues to move through the membrane, leaving the RNA fragments in place. This process is essential for creating an exact imprint of the RNA fragments on the membrane, which is then ready for probing with a specific probe.
🔎 Hybridization and Detection of Target RNA
The final paragraph focuses on the hybridization process and the detection of the target RNA using a specific probe. The speaker clarifies that DNA is commonly used as a probe for RNA due to better interaction properties. Once the RNA fragments are transferred to the membrane, the probe, which can be labeled with radioactive isotopes or chemiluminescent molecules, binds specifically to the target RNA. This binding results in a visual signal, such as light or color, which can be captured on X-ray film to produce an autoradiograph. The autoradiograph reveals the presence and size of the target RNA fragment, allowing researchers to identify the specific RNA of interest. The speaker concludes by encouraging viewers to like, subscribe, and share the video, hinting at upcoming Western blotting videos.
Mindmap
Keywords
💡Northern Blotting
💡Southern Blotting
💡Hybridization
💡Messenger RNA (mRNA)
💡Denaturing Gel Electrophoresis
💡Formaldehyde
💡Agarose Gel
💡Nitrocellulose Filter Paper
💡Aminooxy Methy Paper
💡Probe
💡Autoradiography
Highlights
Introduction to Northern blotting as a technique for detecting RNA, similar to Southern blotting for DNA.
Explanation of the necessity to understand Southern blotting before learning about Northern blotting.
Historical context of blotting techniques, starting with Southern blotting in 1975 and Northern blotting in 1979.
Description of the three major stages in the blotting process: preparation, separation, and detection.
Extraction of RNA as the first step in Northern blotting, focusing on messenger RNA (mRNA).
Use of nucleic acid hybridization to identify a specific sequence of RNA from a mixture.
Generation of complementary DNA strands to serve as probes for RNA detection.
Attachment of radioactive or chemiluminescent molecules to probes for detection purposes.
Utilization of denaturing gel electrophoresis to resolve RNA secondary structures into linear forms.
Difference in gel composition for Northern blotting, including the addition of formaldehyde to linearize RNA.
Transfer of RNA from the gel to a membrane using capillary action in the blotting process.
Use of aminooxy methy paper instead of Nitro cellulose for better RNA binding in Northern blotting.
Setup of the blotting apparatus, including buffer, sponge, gel, membrane, and paper towels.
Process of capillary action transferring RNA from the gel to the membrane filter paper.
Identification of specific RNA fragments of interest using a radioactive or chemiluminescent probe.
Differences between Northern and Southern blotting, including the type of nucleic acid and probe used.
Practical applications of Northern blotting in identifying specific RNA sequences.
Invitation to like, subscribe, and share the video for upcoming Western blotting content.
Transcripts
[Music]
welcome back friends welcome to another
video tutorial from shos biology in this
video lecture we'll be talking about
Northern blotting okay now before
understanding Northern blotting it's
very important that you know Southern
blotting or at least to any of the
blotting techniques because Northern
blotting is a very very similar with
Southern blotting technique so if you
don't know about the southern blotting
technique yet I I'll recommend you to
watch my Southern blotting video which
the description is provided and the link
is provided in the description as well
as uh the link I'll try to put here in
The annotation watch that video first
because there is small modifications of
the Southern blotting in case of
Northern blotting okay so let's see
blotting is a process of
detecting any macro molecules that we
deal with like DNA RNA or proteins if
it's a process of detecting DNA we call
it a southern blotting if it's a process
of detecting RNA we call it Northern
blotting because the names like with the
directions but actually it's nothing to
deal with the directions exactly uh the
first type of blotting technique that
was discovered in
1975 by em Southern based on the name of
the discoverer it was Southern blotting
but later in s in in 1979 4 years later
another technique was discovered uh that
is known as a northern blotting because
you know Southern blotting is a process
of detecting DNA in 1975 it was
discovered in 1979 they discovered the
process of detecting RNA so RNA and DNA
very closely linked so earlier it was
Southern blotting that's why they name
it as a northern blotting when it detect
into the RNA now what is the process of
Northern blotting it's very very similar
that of the Southern blotting in any
blotting process there are three major
stages the first stage is to prepare the
Target the Target in this case of
Northern blotting is RNA so for example
RNA is present inside the cell all the
time in the cytosol of the cell or
inside the nucleus so what we can do
here we simply extract the content of
the cell that should contain RNA
especially we do the process of this
Northern blotting in case of messenger
RNA or mRNA
and the content of mRNA is present
inside the cytool so we take that RNA
out we extract that part of the RNA so
we have a RNA mixture which is a whole
RNA mixture of the cell but again like
any other detection techniques or
blocking techniques the idea is to find
out a specific Target of the RNA which
we know the sequence by the way so how
would you find out a known sequence of
the RNA from a mixture of other rnas the
only way to do that is is do the
hybridization technique or nuclic acid
hybridization because you know nuclic
acid has a very important feature for
example if I tell you the target RNA
that we are looking for has the sequence
a a u u a g g c u a for example this is
the RNA sequence that we want to find
out okay we know that DNA and RNA both
have a compliment feature so if they
find any complimentary strand they are
going to attach to it the complimentary
strand of this RNA will be you know if
we if we uh get this strand in based on
RNA sequence it will be u u a a u c c g
a u if we think of this complimentary
stand as a DNA fragment it could be t t
a a t c c g g a
t so these are the compliment DNA
strands that we have to generate so once
we generate the strands we can put the
Strand as a probe and that can bind to
the Target DNA now if we attach this
this strands this target strands along
with some sort of radio leled or radio
Radioactive molecule or if we attach
with any sort of chemical or enzyme
molecule which is is giving us light
after a reaction we can accurately
identify the position of the target RNA
in the mixture of RNA while we separate
it in the
gel remember that that's what we do in
the northern blotting so the first step
what we do is we extract all the RNA
then we break those rnas down in smaller
fragments because you know rnas tend to
present in complex secondary structure
forms because they are single stranded
so so they they can have the ability to
pair with wherever they find a
complimentary region so RNA never
presents like a linear strands like that
RNA always present in the very complex
structures and secondary structures
formed right we want linear structure of
the RNA and the fragments of linear
structure of the RNA for the process of
nucleic acid probing or nuclic acid
hybridization then only we can find out
the specific sequence of the RNA now if
you look at
here so how can we break this secondary
structure of the RNA down into linear
form so in that case we need to use a
denaturing gel electroforesis process
which will help us to resolve the
secondary structure into linear RNA then
we can separate that RNA using
electroforesis which is an agar Ro gel
electroforesis then we use the probe and
then the process will be done so this
whole stages will be known as the
northern blotting so first what we do so
extraction is done we have a secondary
structure of the
RNA secondary structure of RNA so what
we do now we load this RNA onto agaro
gel let's say this is the agaro gel that
we are talking
about say this is this this drawings are
from the previous lecture that is
Southern blotting which is you you'll
see that very very similar so what we do
we load this thing we load this
secondary structure of the RNA in the
agaro gel now in the agaro gel when we
load them you know agaro gel that we use
here in case of sou Northern blotting is
different compared to the agaro gel we
used in case of Southern blotting in
this agos gel we have
extra formal
deide we add formal deide in the gel
why because this formal deide will help
to resolve the secondary structure into
linear form of the RNA and that is very
very important otherwise uh the RNA will
not be separated based on their length
the idea of using agos gel is to
separate nuclic acid contents based on
their length because agaro is a polymer
of small Matrix and they'll form small
pores inside so smaller length RNA or
DNA fragment can migrate further along
this gel while the larger fragments the
longer fragments will migrate Less in
the gel with the help of this we can
identify where the larger fragment of
the RNA is present where the smaller
fragment of the RN is present but if the
RNA is in secondary structure format
they will not follow this rule so we
need to resolve the structure into
linear form we use formal deide to do
that formal deide makes it linear and
also little bit heat that helps it to
teat and then the RNA will migrate so
once the the aaro gel electroforesis is
complete so we have the bands or the
pattern of where exactly the different
fragments of the RNA present we get this
idea as a band in the agaro gel but the
question is agaro gel is fragile we
cannot use agaro gel for the probing
process because for the probing we need
to apply different Solutions and buffers
weight and all these things that will
not be enough for uh for holding all
those tension by this agoros gel so it's
a better idea to transfer the content of
the gel into a
paper that is usually in case of
Southern blotting was a Nitro cellulose
filter paper now in case of this
Northern blotting we can also use Nitro
cellulose filter paper but it was found
out that nitr cellulose paper is not
working that well in case of Northern
blotting in this case we use aminooxy
methy paper or aminooxy methy filter
paper instead of a Nitro cellulose
filter paper because you know uh at the
beginning we use Nitro cellulose paper
the results are different but then
slowly in search for a better uh
transfer medium we use aminooxy methy
filter paper it's kind of a very simple
that of the Nitro paper or any nylon
paper but it has more affinity and
greater binding Affinity towards the RNA
so what we do now we take this this gel
we take the gel we put it here so now we
do the process of imprinting or
transferring the content of RNA from the
agaro gel to the filter paper okay and
that is when the plotting term comes in
blotting it's the imprinting of a gel
material onto the paper
and that's why we have this whole
process and this blotting technique of
northern and southern is very very
similar because both this technique uses
the pro the the the process of capillary
action for transferring the content of
DNA or RNA from the gel to the membrane
filter now let's look at here in this
total blotting setup in the blotting
setup we have a buffer we have a of a
buffer that that is uh that that will
contain all the mainly it's it's water
containing buffer mostly but we cannot
use plain water for any biological
processes we buffer here and then on top
of buffer we put sponge the sponge helps
there because the water will move sponge
will help in the capillary activity and
up on the top of the sponge we have the
gel so let me let me just highlight the
gel this is the
gel agaro
gel okay and on top of the gel we put
the aminooxy
methy we put the membrane or filter let
me write it down here this is
the the RNA uh filter and we have this
gel the
sponge and on so look at here the gel
and the filter paper are placed on top
of each other first gel then on top that
we have the filter and on the top top of
filter we put some paper towel okay and
then on top of that we put some weight
so this setup is kind of same in case of
Southern blotting as well as Northern
blotting okay now the idea is the the
those buffer will flow based on the
capillary activity from the bottom
towards the top as we apply some weight
from the top according to the capillary
flow water can flow against the
gravitation and it will flow from the
bottom towards the top so as the water
is moving it moves first through the
sponge reaches the gel it passes through
the gel as it's passing through the gel
it is creating Force to all those RNA
fragments that are present there so it
will be pushed and the RNA fragments are
now released from the gel and then they
will migrate towards this filter paper
and once this RNA fragments will go and
attach to the filter paper there is no
way those RNA can migrate because filter
paper is not permeable to RNA molecules
so RNA will be stagnant it will be
attached to the filter paper there while
buffer solution can easily pass through
the membrane and it will reach the paper
towels that's why you put a lot of paper
towel stack there over so that in a
sense of how exactly we push the
fragments of the RNA and and apply it to
attach to the membrane filter once it's
attached to the membrane then the
process is done we exclude everything
out we take the membrane out now the
membrane will have exact imprint of all
those fragments of the RNA that were
present in the gel so now we take that
membrane and remember one thing
importantly because RNA is single
stranded so all these rnas are present
in the in the in the filter paper then
what we do we add the probe which is
designed to go and bind exactly with the
target RNA only the probe is attached
with with radioactive or radiel isotope
or it can be attached with any chem
luminence molecule in either way this
probe is going and bind only with a
specific
Target and that is going to give us
either light or any color or radio if
it's radio leveled isotope then you put
a Xtra film on top of that and after
developing the Xtra film what we'll get
let's say the small version if I draw
this small version of that we will only
find one b and where the target DNA was
present so you'll see by looking at this
gel or or Auto radiogram we can easily
tell which RNA fragment carry our D RNA
of interest in this case let's say this
one if we go back to the gel we can say
yes this is the fragment that carries
the target DNA of our interest so that
is how the target RNA of our interest
sorry that is how the northern blotting
is conducted and it's very similar that
of the Southern blotting the difference
is that in this case we need to use
formal deide in the gel while we're
separating another difference is that uh
in this case of a southern blotting DNA
were attached to the membrane so we need
to denature this another strand to make
it a single strand but in case of RNA we
don't have to do that remember in case
of RNA hybridization or Northern
blotting we need to use we mostly used
DNA as a probe not RNA because RNA RNA
interactions are not that good DNA
interaction with RNA is mostly done so
the the probe with the DNA is simply RNA
DNA
hybrid that we use but in case of
Southern blotting we use DNA DNA
hybridization now the DNA that we
produce here is called complimentary DNA
because it's designed uh by looking at
the complimentary RNA structure okay so
that in a sense is a difference between
southern blotting and western uh
Northern blotting okay so I hope you
understand the video If you like this
video please hit the like button
subscribe to my channel because Western
blotting videos are going to come and
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thank you
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