Measuring mRNA expression

Transcriptome

everything that is transcribed from DNA into RNA

What different types of RNA constitute the transcriptome?

• mRNA (protein coding)

• house-keeping RNAs

• long non-coding RNAs

• small non-coding RNAs

What are examples of house-keeping RNAs?

• ribosomal RNA (rRNA)

• transfer RNA (tRNA)

How long are long non-coding RNAs? 

> 200 nucleotides

How long are small non-coding RNAs?

< 200 nucleotides

What types of RNA have a polyA tail?

• mRNA

• long non-coding RNAs

What is the role of house-keeping RNAs?

involved in translation of mRNAs

What are examples of small non-coding RNAs?

• miRNAs

• snoRNAs

• snRNAs

• piRNAs

What is an exon?

A part of the gene that is transcribed into mRNA

What happens to the exons of the gene once they are transcribed?

they are spliced together to form various transcripts

What are RNA databases? What kind of information do they provide?

A list of gene sequences and their genomic positions:

• refernce genome (e.g. GRCh38/hg38-human)

• position on the chromosome

• which strand it is from

• information on exons, introns, 3’-UTR, 5’-UTR

What does RNA have to be converted to cDNA for sequencing?

because it is very unstable (there are RNAses everywhere)

What is the historic method of measuring mRNA expression levels?

microarrays

What are the steps of a microarray?

• mRNA is converted to cDNA using a reverse transcriptase

• a fluorescent label (e.g. cy3, cy5) is added to cDNA

• labelled cDNA is added to the microarray slide (the slide contains probes complementary to the mRNA of interest, so the target cDNA binds to them)

• everything that is unbound is washed off

• fluorescent signal from bound cDNA is measured to quantify the amount of cDNA/mRNA

How are RNA databases used for microarrays?

to design the microarray probes

How long is a microarray probe?

25-60 nucleotides

How many probes are on a slide?

100s of identical probes are clustered into dots, and 3-12 dots are placed on a microarray slide for each exon or transcript

Why are so many identical probes on a microarray slide?

to prevent saturation at high mRNA concentrations

How are microarray data analysed?

• an average of all the dots for one transcript is taken

• background is subtracted

How is the level of background signal identifies for microarrays?

using a probe with one mismatched nucleotide to represent random binding

What are the different types of RNA sequencing?

short read sequencing (Illumina)

• bulk RNA sequencing

• single cell RNA sequencing

• spacial transcriptomics

long read sequencing (Nanopore)

What is bulk RNA sequencing?

sequencing RNA from a large number of cells from a cell culture or a tissue sample, which provides an average of mRNA across all cells in the sample

What is the main disadvantage of bulk RNA seq?

It can be difficult to interpret data when there are many cell types in a sample

What are the steps in bulk RNA sequencing?

• RNA is isolated form a sample

• RNA is converted to cDNA and amplified if needed

• cDNA is fragmented (sonicated or digested with enzymes)

• a library is prepared

• Illumina sequencing

• data analysis

Why does cDNA need to be fragmented for bulk RNA sequencing?

the Illumina sequencer can only sequence 600 nucleotides at a time, but RNA is ~3000 nucleotides on average

What is Illumina sequencing?

sequencing by synthesis

• fluorescently tagged nucleotides are used

• after the addition of each nucleotide, the sample is exited, and a characteristic fluorescent signal is emitted

How is data from but RNA sequencing analysed?

• the short reads (in the form of FASTQ files) are aligned against exons on a reference genome using a programme (e.g. Hisat2)

• all the reads that align at the same exon are summed up

• the number of reads is proportional to the level of mRNA expression

How are RNA databases used in short read RNA sequencing?

to identify the position of genes (exons) on a reference genome in order to align the sequencing reads against them

What is single cell RNA sequencing?

individual cells are isolated from a sample, and mRNA expression within one cell is measured

What is the most common way to isolate cells for single cell RNA seq?

microfluidics (e.g 10X genomics)

How does 10X genomics work?

• beads are coated with probes

• the beads flow through one channel of the microfluidics machine, the cells flow through a different channel of the microfluidics machine

• the flowing beads and cells interact with a continuous oil phase

• oil droplets are formed, containing one bead and one cell in each

• the cell is lysed within the droplet, and the bead collects all the mRNA from the cell

What do the probes on beads for single cell RNA seq consist of?

• PCR handle

• cell barcode (to identify which cell the mRNA is from)

• unique molecular identifier (allows to determine the number of mRNAs by aligning to the reference genome)

• a TTTTTTTTTT segment

What are the advantages of single cell RNA seq compared to bulk RNA seq?

• allows to look at individual cells

• allows to identify different populations

What are the disadvantages of single cell RNA seq compared to bulk RNA seq?

• individual cells need to be isolated

• only detects the most expressed genes

• bioinformatics is more complex

• more expensive

What is spacial transcriptomics?

allows spacial visualisation of mRNA expression:

• beads with probes are placed on a slide

• tissue is placed on top

• cells are lysed, and mRNA binds to the bead

How does long read sequencing work?

• an adapter is attached to the sequence

• the sequence is pulled through a pore

• the disruption of ionic currents as nucleotides go through is measured to identify the nucleotide

What is long read sequencing mostly used for?

to identify bacteria for diagnostic purposes

What are the advantages of long read sequencing over short read sequencing?

• can read the whole sequence in one go

• doesn’t require conversion of RNA to cDNA

• quick

• cheap to set up

What are the disadvantages of long read sequencing over short read sequencing? And why is it not commonly used for RNA sequencing?

• can only read a a small number of sequences (not very efficient for processing large amount of RNA)

• an error rate of 1% (to high of an error rate if looking for a SNP)

What are the advantages of RNA sequencing compared to microarrays?

• can detect RNA transcribed from an unknown gene

• allows single cell and spacial transcriptomics

• better repeatability (samples can be run on separate occasions and still compared)

• larger quantification range (high and low concentrations of RNA)

• allows detection of SNPs and splice variants

What are the disadvantages of RNA sequencing compared to microarrays?

• bioinformatics expertise required

• uses large amount of computing power and storage

What is important to bear in mind with any RNA level detection method?

• must be confirmed using an alternative technique

• there isn’t necessarily a correlation between amount of RNA and amount of protein

What technique can be used to confirmed the results of RNA sequencing?

Real-time PCR