Lecture 3 - Gene expression analysis

Dr Federico Rojas
[email protected]
 Learning
objectives

After attending this lecture and carrying out the recommended reading you should be able to:

- List the three parameters of gene expression

- Describe different kinds of nucleic acid probe
- Describe methods for measuring mRNA levels
- Explain what a microarray is
- Explain what is meant by the transcriptome
- Describe how can you determine epigenetic changes

Reading: Campbell and Reece 10th ed., pages 457-462

 Gene expression analysis
Analysis of when and where a gene or group of genes is expressed can provide important clues about their
function and how they contribute to the organism as a whole.

What do we want to analyse?

1. mRNA
2. Protein

What do we want to know about gene expression?

1. Level (relative or absolute)

2. Time (during development or in response to external factors eg.
Drugs, hormones)
3. Place (organ, tissue, cell, subcellular)

Eukaryotic cell.
 Gene expression analysis

Techniques for measuring mRNA levels

- Northern blotting
- RT-PCR
- Microarray (GeneChip) techniques

Techniques for measuring protein levels

- Western blotting
Many techniques for measuring nucleic acid levels require probes

Radioactive (detected by autoradiography)

–32P : high energy, sensitive
–35S, 14C, 3H : lower energies, less sensitive, higher resolution

Chemical
–“antigen” incorporated into DNA, bound by enzyme-linked antibody, makes a coloured
or chemiluminescent compound

Fluorescent
–Many different fluorescent compounds are available
–Detect with specialised equipment
 Reverse Transcriptase PCR (RT-PCR)

RT-PCR uses the enzyme reverse transcriptase (RT) in combination with PCR and gel electrophoresis.

RT-PCR can be used to compare gene expression between samples:

i.e. in different embryonic stages, in different tissues, or in the same type of cell under different conditions.
 Reverse Transcriptase PCR (RT-
PCR)
Reverse transcriptase is added to a test tube containing
mRNAs, isolated from a sample of cells.

Reverse transcriptase makes the first DNA strand using the mRNA
as a template and a short poly-dT as a DNA primer.

mRNA is degraded by RNAse H

DNA polymerase synthesizes the second DNA strand, using a primer in

the reaction mixture

The result is cDNA, which carries the complete coding

sequence of the gene but no introns.
Reverse Transcriptase PCR (RT-PCR)

Example: Samples containing mRNAs from six 1. cDNA synthesis

embryonic stages of Drosophila were analyzed
for a specific mRNA.
2. PCR amplification

Results: The mRNA for this gene is expressed 3. Gel electrophoresis

from stage 2 through stage 6.
 Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene
Expression

Automation has allowed scientists to measure the

expression of thousands of genes at one time using
DNA microarray assays

DNA microarray assays compare patterns of gene

expression in different tissues, at different times, or
under different conditions

Each microscopic spot on the microarray is a 50-

nucleotide DNA molecule of a defined sequence
made by chemical synthesis and spotted on the
array.
 Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene
Expression

DNA microarrays are used to analyze the production

of thousands of different mRNAs in a single
experiment

The DNA sequence represented by each spot is

different, and the hundreds of thousands of such spots
are designed to span the sequence of the genome.
 Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene
Expression
Researchers extracted mRNAs from two different human tissues and synthesised two sets of cDNAs;
fluorescently labelled Red (tissue 1) or Green (tissue 2)

Labeled cDNAs were hybridized with a microarray containing 5,760 human genes (25% genome).

Genes that were expressed more in Tissue 1

Genes that were expressed more in Tissue 2

Genes that were expressed in both tissues, these

wells appear yellow.

Genes that were not expressed in either tissue;

these wells appear black
 Use of RNA sequencing (RNA-seq) to analyse expression of many genes.

Possible to study the expression of large groups of genes

Today’s rapid, inexpensive DNA-sequencing methods allow

researchers to discover which genes are expressed by simply
sequencing the cDNA samples from different tissues or different
embryonic stages.

Short sequences
are mapped by
computer onto the
genome sequence
 Using cluster analysis to identify sets of genes that are coordinately
regulated.

Genes that have the same expression pattern are likely to be involved in common pathways or processes.

RNA-seq or microarray data are obtained from cell samples exposed to a variety of different conditions
genes that show coordinate changes in their expression pattern are grouped together.
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 In Situ Hybridisation (ISH)

Determining where single genes are expressed by

in situ hybridisation analysis.

Time and location i.e the distribution of mRNA in

a whole organism, tissue or cell!

Drosophila embryo incubated with DNA probes for five

different mRNAs, each probe labelled with a different
fluorescently coloured tag
 Epigenetics

Heritable changes in gene activity that are not the result of changes in DNA sequence
This can lead to changes in gene expression and cellular phenotype

How does epigenetics work?

In the nucleus, DNA coils and condenses around histones.
The DNA–protein complex is referred to as chromatin.

The addition of chemical groups to the DNA (Methylation) or

the histone proteins in the chromatin determines whether or not
a gene is expressed.
 Epigenetics

How do determine if a change/difference in gene expression is the result of an epigenetic change?

(Examining the DNA sequence will not tell us anything- will it?)
 3rd Generation Sequencing

Third generation sequencing technologies offer the capability for single molecule real-time sequencing of longer
reads, and detection of DNA modification.

1. PACBio
A T C mC

DNA is fragmented and ligated Fluorescently labelled The light emitted from the
to adapters deoxynucleoside excited fluorophore is detected
+ triphosphates (dNTPs) are by a camera.
DNA polymerase added Records the wavelength and
(Cell can contain up to 8 relative position of the
incorporated base in the nascent
million chambers) strand
 2. Oxford Nanopore

A T G C mC

2. Motor protein begins to unwind the 3. DNA moves through the pore, it
1. The flow cell contains thousands of
double-stranded DNA. causes characteristic disruptions to
protein nanopores embedded in a synthetic
Electric current is applied, driving the the current.
membrane, and the tethering proteins bring
negatively charged DNA through the Each nucleotide has a specific
the DNA molecules towards these nanopores
pore “signature”

Long-read human genome sequencing and its applications. Nature Reviews Genetics. 597–614 (2020)
Introduction to nanopore sequencing
video
youtube.com/watch?v=qzusVw4Dp8w
 Why is important to determine epigenetic
modifications?

• In 2011, an E.coli outbreak in Germany affected thousands of people.

50 died and nearly 1000 people suffered kidney failure.
• Sequencing found that the methylation pattern, but not the DNA sequence had changed.
• This probably contributed to the pathogenicity of what was previously thought to be a non-
pathogenic strain

https://www.pacb.com/wp-content/uploads/2015/09/CS_Beyond_Four_Bases.pdf