RNA sequencing
Summary
TLDRThis video delves into the significance of RNA sequencing, a critical yet often overlooked aspect of transcriptomics. It explains the concept of the transcriptome, encompassing all RNA within a cell, and the importance of RNA sequencing in detecting mutations, alternative splicing, and post-transcriptional modifications. The video outlines two primary RNA sequencing methods: direct and indirect, with a focus on mRNA isolation via poly-A library for direct sequencing and the creation of cDNA for the indirect approach. It also touches on the challenges of RNA's fragility and the prevalence of DNA sequencing facilities, advocating for the direct method when possible.
Takeaways
- π¬ RNA sequencing is a crucial technique in transcriptomics, allowing researchers to understand the cell's RNA content comprehensively.
- 𧬠The transcriptome encompasses all RNA present in a cell, including mRNA, tRNA, non-coding RNA, and degraded RNA fragments.
- π RNA sequencing can reveal single nucleotide polymorphisms (SNPs) and mutations that occur during the transcription process from DNA to RNA.
- π It can also detect alternative splicing, which is the process of producing multiple varieties of mRNA from a single gene, leading to diverse protein functionalities.
- π Post-transcriptional modifications and gene fusions are other cellular features that can be identified through RNA sequencing.
- βοΈ RNA is more fragile than DNA due to the absence of a 2'-OH group in its sugar molecule, making its handling and sequencing more challenging.
- π§ͺ Two primary methods of RNA sequencing are direct RNA sequencing and indirect RNA sequencing, which involves first converting RNA to complementary DNA (cDNA).
- π Isolation of specific types of RNA, such as mRNA via poly-A selection, is a critical step before sequencing, as different RNA types require different isolation techniques.
- 𧲠Techniques like size exclusion chromatography and magnetic bead systems are used for isolating smaller RNA fragments, such as tRNA and degraded mRNA.
- 𧬠High-throughput or next-generation sequencing technologies are commonly employed for RNA sequencing to analyze the vast amount of RNA data efficiently.
- π Direct RNA sequencing is preferred for its accuracy, but indirect sequencing through cDNA is more accessible due to the widespread availability of DNA sequencing technologies.
Q & A
What is RNA sequencing?
-RNA sequencing is a process that involves sequencing all the RNA content present in a cell, including mRNA, tRNA, non-coding RNA, and degraded RNA, to obtain a detailed picture of the cell's transcriptome.
Why is RNA sequencing important in transcriptomics?
-RNA sequencing is crucial in transcriptomics because it provides insights into the cell's RNA content, which can reveal single nucleotide polymorphisms, mutations, alternative splicing, post-transcriptional modifications, and gene fusions.
What is the difference between DNA sequencing and RNA sequencing?
-DNA sequencing focuses on the order of nucleotides in DNA, while RNA sequencing focuses on the RNA molecules transcribed from DNA, which can include post-transcriptional modifications and alternative splicing events not present in the DNA.
What is the transcriptome?
-The transcriptome refers to the complete set of RNA molecules, including mRNA, tRNA, rRNA, and non-coding RNA, present in a cell at a given time.
How can RNA sequencing detect single nucleotide polymorphisms (SNPs)?
-RNA sequencing can detect SNPs by identifying variations in the RNA sequence that differ from the DNA template, which may occur during the transcription process.
What is alternative splicing and how can it be detected through RNA sequencing?
-Alternative splicing is a process where different mRNA transcripts are produced from the same gene, leading to the production of multiple protein variants. RNA sequencing can detect alternative splicing by identifying different mRNA variants from a single gene.
Why is RNA more challenging to sequence compared to DNA?
-RNA is more challenging to sequence because it is more fragile and prone to degradation due to the absence of the 2'-OH group in its ribose sugar, which makes it less stable than DNA.
What is the poly(A) library method used for in RNA sequencing?
-The poly(A) library method is used to isolate mRNA from a mixture of RNAs in a cell because mRNAs have a poly(A) tail at their 3' end, which can bind to oligo(dT) beads, allowing for the enrichment of mRNA for sequencing.
What are the two main types of RNA sequencing mentioned in the script?
-The two main types of RNA sequencing mentioned are direct RNA sequencing, which sequences the RNA molecules directly, and indirect RNA sequencing, which involves first converting RNA into complementary DNA (cDNA) and then sequencing the DNA.
Why is reverse transcription used in RNA sequencing?
-Reverse transcription is used to convert RNA into complementary DNA (cDNA), which is more stable and easier to sequence. This process allows for the use of widely available DNA sequencing technologies to determine the RNA sequence information.
What is the significance of the next-generation sequencing (NGS) in RNA sequencing?
-Next-generation sequencing (NGS) is significant in RNA sequencing because it allows for high-throughput, rapid, and cost-effective sequencing of RNA molecules, enabling the analysis of the transcriptome at an unprecedented scale.
Outlines
𧬠Introduction to RNA Sequencing
The video introduces RNA sequencing, a process that is often overshadowed by DNA sequencing but is crucial for understanding the cell's transcriptome. Transcriptome refers to the complete set of RNA, including mRNA, tRNA, non-coding RNA, and degraded RNA, present within a cell. RNA sequencing, also known as whole transcriptome shotgun sequencing (WTSS), is used to sequence every piece of RNA to gain insights into the cell's functions. The importance of RNA sequencing lies in its ability to detect single nucleotide polymorphisms (SNPs), mutations, alternative splicing, post-transcriptional modifications, and gene fusions. These insights are vital for understanding cellular processes and variations that may not be evident from DNA alone. The video also touches on the challenges of RNA sequencing due to the fragile nature of RNA and the two main types of RNA sequencing: direct and indirect.
π¬ RNA Extraction and Isolation Methods
This section delves into the technical aspects of RNA sequencing, starting with the extraction of RNA from cells. It emphasizes the importance of isolating specific types of RNA, such as mRNA, tRNA, and small RNAs, due to their diversity and abundance within the cell. The video explains the use of poly-A libraries for mRNA isolation, which leverages the poly-A tail present in mature mRNA to bind to poly-T beads, thus separating it from other types of RNA. The video also mentions alternative isolation techniques like size exclusion chromatography and magnetic beads, which are suitable for smaller RNA fragments. The process of creating a poly-A library is described, where mRNA is selectively bound to beads, creating a collection enriched with mRNA sequences ready for sequencing.
π§ͺ High-Throughput Sequencing Techniques
The final paragraph discusses the sequencing process itself, highlighting the use of high-throughput or next-generation sequencing (NGS) technologies. These advanced techniques allow for the rapid sequencing of large numbers of RNA molecules. The video differentiates between direct and indirect RNA sequencing methods. In direct RNA sequencing, the RNA is sequenced without the need for reverse transcription to DNA. In contrast, indirect RNA sequencing involves converting RNA to complementary DNA (cDNA) using reverse transcriptase and then sequencing the DNA. The video points out that while direct sequencing is more accurate, indirect sequencing is more accessible due to the widespread availability of DNA sequencing technologies. It concludes by emphasizing the importance of RNA sequencing in genomics and the potential for error in the indirect method, suggesting that direct sequencing is preferable when possible.
Mindmap
Keywords
π‘RNA sequencing
π‘Transcriptome
π‘Single nucleotide polymorphism (SNP)
π‘Alternative splicing
π‘Post-transcriptional modification
π‘Gene fusion
π‘Poly A library
π‘Size exclusion chromatography
π‘Reverse transcription
π‘Complementary DNA (cDNA)
Highlights
RNA sequencing is crucial for understanding the transcriptome, which includes all RNA content in a cell.
Transcriptome analysis is essential for studying gene expression at the RNA level.
RNA sequencing can detect single nucleotide polymorphisms and mutations in RNA that occur during transcription.
Alternative splicing, which generates diverse mRNA transcripts from a single gene, can be identified through RNA sequencing.
Post-transcriptional modifications and gene fusions are detectable through RNA sequencing, providing insights into gene regulation.
RNA is more fragile than DNA due to the absence of a 2'-OH group, making it challenging to handle.
There are two main types of RNA sequencing: direct and indirect, each with its own advantages and challenges.
Direct RNA sequencing involves sequencing the RNA molecule without converting it to DNA.
Indirect RNA sequencing uses reverse transcriptase to first convert RNA to complementary DNA (cDNA) before sequencing.
Poly A library is a common method for isolating mRNA from the total RNA content in a cell.
Size exclusion chromatography and magnetic beads are techniques used for isolating smaller RNA fragments.
High-throughput or next-generation sequencing is typically used for RNA sequencing due to its efficiency.
Understanding the differences between direct and indirect RNA sequencing is key to choosing the appropriate method for research.
RNA sequencing provides a detailed picture of the cell's transcriptome, aiding in the study of gene expression and regulation.
The choice between direct and indirect RNA sequencing depends on the availability of RNA sequencing facilities and the research goals.
RNA sequencing can reveal important insights into cellular processes, including gene expression and regulation.
Transcripts
welcome again guys in this video we'll
be talking about RNA sequencing RNA
sequencing is something that people
don't talk about too much people talk
about DNA sequencing but RNA sequencing
is also important and nowadays once in
the zone of transcriptomics we lot of
the time we need to sequence RNA to
chick certain important feature inside
the cell so that is usually called as
the the whole transcriptome whole
transcriptome RNA sequencing RNA shotgun
sequencing simply w-t is is whole
transcriptome shotgun sequencing so what
is transcriptome we have already talked
about it if you don't know what it is
you can go back to my channel you'll
find a video about transcriptome watch
that video and then you can join us but
actually transcriptome means all the
content of RNA that is present in a cell
every bit of RNA mRNA tRNA our RNA a
non-coding part of the RNA degraded part
of the RNA whatever RNA content is
present inside the cell all together
is termed as transcriptome so if we need
to sequence all of them we take all the
transcriptome of the cell and then we
sequence every single piece of RNA small
RNA degraded RNA longer RNA every bit of
RNA to get the detailed picture of our
any inside the cell now the question
remains sequencing genome is important
and we know that but what is the
importance of RNA sequencing now the
important is that RNA sequencing can
tell a certain site important insight
about the cell why is that one of the
important thing about RNA sequencing is
that RNA sequencing can give us the idea
about any kind of single nucleotide
polymorphism or mutation that is found
in the RNA content say from the DNA we
produce RNA but during the production of
RNA from the DNA's in transcription
process there might be some changes some
mutation
some single nucleotide polymorphism that
may reserve in change the in the in the
sequence of RNA and we can detect it
using the SNP mutation this this RNA
sequencing process that is one thing
that we can detect about Dharan
sequencing another important thing that
we can detect is that there is is there
any change any kind of change here so
that is one thing and other features
also includes the alternative splicing
alternative splicing if you don't know
what it is I have a separate video about
alternative splicing also you must watch
that video alternative splicing is like
the other type of splicing which varies
and changes different variety of mRNA
transcript inside the cell from one
single gene it will produce multiple
varieties of mRNA inside the cell so
from one gene we can produce many
different varieties of proteins inside
the cell with different functionality
that is alternative splicing so we can
detect if the cell undergoes any kind of
alternative splicing or not if we have
any SNP or mutation or not or is their
presence of any post transcriptional
post-transcriptional modification so we
can detect all these processes SNP
mutation or alternative splicing or
post-transcriptional modification gene
fusion is another thing we can detect
using RNA sequencing technology so
that's why we need to sequence RNA now
how could we sequence RNA now dealing
with RNA is much more difficult than
dealing with DNA because RNA is very
much fragile it is vulnerable to damage
because you do not have that to prime or
H present in the ribose sugar which is
present in deoxyribose that creates a
very important inside there and have h
there
instead of simple simple H so that is
the problem there so anything now this D
now this RNA sequencing relies in two
different ways there are two different
types of RNA sequencing actually if I if
I like the types two different types are
present one is the the direct RNA
sequencing another one is the indirect
RNA sequencing in either way some basic
characteristics remains the same but
certain things are different so if I
draw the schematic presentation of how
the sequencing will work we know the
first thing is the extraction of RNA
using the normal RNA extraction method
from a cell then isolation of RNA
content because the RNA content is huge
normally the DNA is kind of same in all
the cells the DNA contents are same but
RNA in itself inside the cell is at
least C of different kinds mRNA our RNA
tRNA if there is any other degraded
portion of the RNA it will be small RNA
small nuclear RNA ragΓΉ nuclear RNA so
all these different varieties of RNA
will be present there so there are
variety of RNA present and it is very
important to isolate the type of RNA we
need to sequence so that is the second
important phase isolation and after the
isolation what we go for we go for the
sequencing process so that is the third
and the final step now this is common
for direct as well as easy rate type of
RNA sequencing now extraction is same in
all these cases I am NOT going to talk
about that but isolation can be done and
achieved by two different ways actually
three different ways here the isolation
for directing detect both these things
if the isolation is only for the mRNA
what we can use we we can use which is
called a poly a library we can use poly
a library if you are only selecting mRNA
and because most of the time we sequence
a mRNA because mRNA is the actual
transcript from where the protein are
made proteins are made right so for that
reason most of the time we need to
sequence mRNA not the other
type of ironies so that's why Polly a
library is the most common process of
isolation of mRNA from from the content
of other RNA mixture but other
techniques lies like there are two other
techniques present here also one
technique is the size exclusion
chromatography another one is the size
exclusion magnetic bead these two
processes are their size exclusion
magnetic beads and size exclusion
chromatography now remember both of
these techniques are there for the
smaller fragment of RNA like small rnas
like tRNA is like other degraded portion
of the mRNAs and all the other types of
RNAs but for the mRNA we use poly a
library now what does that mean what is
poly a library and how does actually
work the poly library is extremely
simple the thing is here I am going to
talk about most of this about the poly a
library process on how this work is that
we have now let us say we have a mixture
so let let me create some of this stuff
over there what is poly a library poly a
library remember in eukaryotic cell
because we are talking about eukaryotic
cell in in details about the RNA
sequencing all this thing's
transcriptome usually talk about
eukaryotes here so in eukaryotes what we
know is that after the amount is
produced that a mine is termed as the
pre mRNA it is treated the five prime
capping C prime poly a realization
should take place to make it as a
complex to make it a mature mRNA so once
the mature mRNA is bid the mature Amanda
should have five prime cap it has a poly
a tail at the three prime so this is the
construct of mRNA tRNA and rRNA do not
have this content so if I draw a tRNA
structure or and let's say the rRNA is a
huge this structure they don't have
those poly a tail now
once we add we have beads if say we have
beads and that bees are having poly T
residues at
to them Poli T residues attached to the
terminal so what is the idea now
so this beats the filled with quality
residues protruding out from the beads
but all of this so they can easily pair
with adenine because timing can pair
without anything but as tRNA and rRNA do
not have any of this poly a tail
they will not bind with the bead but
poly a tail is presenting mRNA
so all these mRNAs can pair with with
this beads so once they pair with the
bead so we can select the specific type
of mRNA only from other varieties of RNA
present in the cell and and once if you
provide once if once we do this task of
isolation using the beads what we call
it as a poly a library because it is a
kind of library filled with all the mRNA
content of the cell and they are added
with the beads so it's kind of poly a
library and as we use poly a tail to
create the library we call it a poly a
library so once the poly library is made
then what we do we use the normal
sequencing process and the sequencing
process for actual sequencing that we
use in this case remember in this case
we use high throughput sequencing or
next-generation sequencing we use this
type of sequencing techniques for that
now what is next-generation sequencing
what is high-throughput sequencing I
will highly encourage you to go and
watch these videos in my channel you
will find video on high-throughput
sequencing you will find the video on
this NGS or next-generation sequencing
in my channel so watch those videos I am
NOT going to talk about them because
there are larger videos so then finally
we sequence it with any of this
processes the next generation of
high-throughput sequencing now this is
the way now now about the direct and
indirect stuff that we are going to talk
about here the directed injects stuff
that is present here that is that is
important thing that sometimes what we
do we only sequence this mRNA let us say
here in this case we
and this mRNA to the beats and then you
sequence is mRNA content only that
nuclei will ribose sugar content here
from only but in other cases that is
called the direct type of sequencing
because we are sequencing directly the
RNA with sequencing the RNA directly so
this is direct sequencing but there are
some times when we do not sequence RNA
what we do we take that RNA we isolate
the RNA that is a very very important
step we isolate that RNA by either a
size exclusion chromatography or
magnetic chromatography magnetic bead a
bit system so the size exclusion
chromatography means simply this is a
chromatography or gel not actually from
sometimes chromatography or gel it is a
gel where the larger fragments trap
smaller fragments travel past so we can
pick up those RNAs from there so once we
have them we do not directly sequence
the RNA what we do so we have the RNA we
build the other strand of DNA the
complimentary strand of the DNA using a
reverse transcriptase enzyme you know we
can produce DNA strand from RNA is in
the reverse transcriptase so if we have
the RNA here we add the
deoxyribonucleotide sequences one after
another and we prepare this
single-stranded DNA sequence there we
produce that deal once you produce that
DNA then we produce the the
double-stranded DNA sometimes sometimes
not if we require or not but that is
called as a complimentary DNA or cDNA
because it is produced from its
complementary RNA strand so that is the
complimentary DNA so sometimes you
produce this DNA using reverse
transcriptase enzyme and the process
called reverse transcriptase the reverse
transcription once you produce that DNA
then we go for the sequencing and that
process is called as the indirect
process of RNA sequencing now you may
ask in what sense it is an RNA
sequencing the answer is there are we
have you know the answer here is that
whatever this is the better thing about
the genomics because whatever thing is
present in the RNA
we know the complementary strand of that
whatever it is DNA or not whatever thing
is present in the complementary we can
actually get the idea the rivet is a the
complementary will be T if it is u the
complementary will be a if it is G in
the RNA the complementary of DNA will be
C so if we know the sequence of DNA we
can count it we're using complementary
nature of the DNA to find out the actual
RNA sequence so we are actually doing it
similar and going back so you may ask
that why we are doing this why when we
are going direct because did it is based
obviously directive is the best DX is
obviously the better technique but what
happens actually the RNA I have told you
RNA handling is difficult and all the
labs RNA sequencing facilities are not
available but DNA sequencing is much
more common so we have machines for the
DNA sequencing rapidly all the time
going on so what we do we we reverse
transcribe their RNA to DNA we do that
we sequence that then we go back track
using the complementary nature of the
rna-dna hybrid and check for the RNA
sequencing but still there are the
chances of error in this compliment in
this indirect type of RNA sequencing so
delayed is obviously the better choice
so that is all about the RNA sequencing
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