RNA sequencing

Summary

This 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

• 🔬 RNA sequencing is a crucial technique in transcriptomics, allowing researchers to understand the cell's RNA content comprehensively.
• 🧬 The transcriptome encompasses all RNA present in a cell, including mRNA, tRNA, non-coding RNA, and degraded RNA fragments.
• 🔍 RNA sequencing can reveal single nucleotide polymorphisms (SNPs) and mutations that occur during the transcription process from DNA to RNA.
• 🌟 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.
• 🔎 Post-transcriptional modifications and gene fusions are other cellular features that can be identified through RNA sequencing.
• ⚖️ 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.
• 🧪 Two primary methods of RNA sequencing are direct RNA sequencing and indirect RNA sequencing, which involves first converting RNA to complementary DNA (cDNA).
• 📚 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.
• 🧲 Techniques like size exclusion chromatography and magnetic bead systems are used for isolating smaller RNA fragments, such as tRNA and degraded mRNA.
• 🧬 High-throughput or next-generation sequencing technologies are commonly employed for RNA sequencing to analyze the vast amount of RNA data efficiently.
• 📈 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

00:00

🧬 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.

05:02

🔬 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.

10:03

🧪 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.

Keywords

💡RNA sequencing

RNA sequencing is a process that involves determining the sequence of RNA molecules in a sample. It's a crucial technique in transcriptomics, which is the study of the entire set of RNA transcripts produced by the genome. In the video, RNA sequencing is highlighted as a method to gain insights into the cell's activities, including identifying mutations and alternative splicing events. It's also distinguished from DNA sequencing, emphasizing its importance in understanding gene expression and regulation.

💡Transcriptome

The transcriptome refers to the complete set of RNA molecules, including mRNA, tRNA, and other non-coding RNA produced in one or a population of cells. In the video, the transcriptome is described as the entirety of RNA content within a cell, which is sequenced to understand the cell's functional state. The video emphasizes that sequencing the transcriptome provides a detailed picture of cellular processes.

💡Single nucleotide polymorphism (SNP)

A SNP is a variation at a single position in a DNA sequence among individuals. In the context of the video, SNPs in RNA are mentioned as one of the key insights RNA sequencing can provide. The video explains that during transcription, changes may occur that result in SNPs, which can alter the RNA sequence and potentially affect protein function, making the detection of such variations significant.

💡Alternative splicing

Alternative splicing is a process by which different combinations of exons (coding regions of genes) are joined together to produce different mRNA transcripts from the same gene. This leads to the production of multiple proteins from a single gene. The video discusses how RNA sequencing can detect alternative splicing events, which are crucial for understanding the diversity of proteins that can be produced by a cell.

💡Post-transcriptional modification

Post-transcriptional modifications are chemical changes made to RNA molecules after transcription. These modifications can affect the stability, localization, and translation of RNA into proteins. The video mentions that RNA sequencing can detect such modifications, which are important for understanding gene expression regulation.

💡Gene fusion

Gene fusion occurs when two separate genes are joined together to form a single gene, often as a result of chromosomal rearrangements. The video highlights that RNA sequencing can detect gene fusions, which can lead to the production of abnormal proteins and are often associated with diseases like cancer.

💡Poly A library

A Poly A library is a method used in RNA sequencing to isolate mRNA molecules, which have a poly A (adenine) tail at their 3' end. The video explains that this technique is commonly used because mRNA is the primary type of RNA that is translated into proteins. The process involves using beads with poly T residues to bind to the poly A tail of mRNA, allowing for its isolation from other types of RNA.

💡Size exclusion chromatography

Size exclusion chromatography is a method used to separate molecules based on their size. In the context of RNA sequencing, as mentioned in the video, this technique can be used to isolate smaller RNA fragments, such as tRNA or degraded mRNA, from a mixture of different RNA types.

💡Reverse transcription

Reverse transcription is the process of synthesizing DNA from an RNA template, using the enzyme reverse transcriptase. The video describes how, in indirect RNA sequencing, RNA is first reverse transcribed into complementary DNA (cDNA), which is then sequenced. This method is used because DNA sequencing is more common and technically easier than direct RNA sequencing.

💡Complementary DNA (cDNA)

cDNA is a DNA copy of an RNA molecule, created through reverse transcription. The video explains that cDNA is produced from the RNA template, which is then used for sequencing. The process allows researchers to leverage the more established DNA sequencing technologies to study RNA molecules.

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 ragù 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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