Experimental Cell Biology II: Single-cell gene expression
There are about 200 different cell types in the This technique relies on hybridization. The RNA
human body that all have been differentiated (or cDNA) extracted from the cells is hybridized
from a stem cell, so they all have the same on specific chips that contain complementary
DNA. The difference between all these cell RNA probes that you expect to see in specific
types can be explained by the differences in organisms. The intensity of the colours will
gene expression. indicate the amount of RNA/cDNA hybridization.
mRNA RNA-sequencing
mRNA is the molecule that transfers the RNA is first transcribed into cDNA. The library of
information from DNA to the protein. It cDNA can then be sequenced and aligned to the
participates in translating the genotype into a genome to measure gene expression.
phenotype and is the key determinant of
protein abundance. Changes in mRNA But, these techniques miss important
expression allow the cell to adapt to changing information about cell-to-cell heterogeneity and
environments by responding to stimuli. spatial information (sub-cellular or within a
Several mechanisms control mRNA/proteins, tissue/organism. It is important to study gene
such as mRNA splicing and posttranslational expression in single cells since every cell has a
modifications. different expression, even when you compare
them to the same cell type in the same tissue. The
How do we study mRNA expression? bulk analysis uses a lot of cells, which could give
This is mainly done through bulk analysis, the wrong idea because the cells of interest may
such as Low-to-mid-plex techniques that be in the minority and their behaviour could get
measure 1-10 mRNAs (northern blot, RT-PCR masked or the dynamics of the populations of
and qPCR) and higher-plex techniques that interest are offset in time.
measure more than 10 mRNAs (DNA
microarray and RNA sequencing).
Northern blot
With the Northern blot RNA, molecules can be
extracted from cells and run on an agarose gel.
The RNA molecules will be separated based on
their size. Fluorescence or radioactivity can be
used to help detect the RNA of interest.
Complementary probe hybridization allows
the measurement of the intensity and size of
the RNA.
Researchers used a population of E.coli and put
RT-PCR/qPCR two genes encoding for fluorescent proteins (GFP
RNA is extracted and with the help of reverse and RFP) under the control of two promoters
transcriptase converted into copy DNA (cDNA, with the same sequence. To their surprise, the
which only contains exons). cDNA can be used population of E. coli showed a lot of variability of
as a template for the PCR. DNA is amplified in colours. The same thing was tested in S.
the presence of dyes such as SybrGreen, which cerevisiae. These results show how random
only lights up when it’s bound to double- events can determine which genes/proteins are
stranded DNA. The increase in fluorescence expressed.
can be detected and RNA can be quantified.
DNA microarray
, But when is it important to study gene and mRNAs (it fixes them and blocks
expression in single cells? degradation). The next step is in situ
This type of study is very important in cancer hybridization where the fixed cells are labelled
research. In tumours, a subpopulation of with isotopic probes. The last step is the detection
cancer cells can develop that have different where the cells are covered with a photographic
capabilities. Some can metastasize and others emulsion. After this, the cells can be observed
are perhaps resistant to certain drugs. By through a light or electron microscope.
studying single cells it is possible to direct
certain medications to those cancer cells. It
could also help to eliminate cells that are on
the path of transforming into cancer cells to
prevent it.
Single-cell studies are also beneficial for the
development of antibiotics since bacteria are
becoming more resistant. In the beginning, a
few bacteria in the culture are resistant. But
following random natural mutations, more The right picture shows the first autoradiograph
bacteria are becoming resistant and can of chicken fibroblast cells hybridized in situ with
survive the antibiotics. 3H-labelled probes to β-actin mRNAs encoding
the cytoskeletal protein actin. This picture shows
Single-cell gene expression analysis methods that RNAs are not localized everywhere in the
cell but are clustered in different cell
compartments.
There are limitations when it comes to using
radioactive probes, such as time and resolution. It
takes weeks to months for the photographic
emulsion to be exposed to detect a signal. There is
also radioactive (3H) decay that causes the
spreading of the signal onto the photographic
emulsion. This makes the localization of the
High-throughput single-cell RNA analysis source inaccurate (µm away).
methods
These techniques are expensive and not Fluorescent in situ hybridization (FISH)
sensitive to molecules at a low concentration. A new approach to allow signal isolation is
• Single-cell RNA sequencing fluorescent probes. These have a higher
• Single-cell proteomics coupled to mass spec resolution and multiple colours are available. The
advantage of this technique is that you can decide
Low throughput single-cell RNA analysis where to excite the fluorophores, which allows
methods you to collect the light from that specific focal
Low-throughput methods are not expensive plane and narrows the resolution down to about
and can find low concentrations of molecules. 200 to 300 nm. The picture shows mouse
• Single-cell RNA imaging with fluorescent chromosomes infected with the EB virus (EBV).
proteins The yellow fluorescent spots on each sister
chromatid of the chromosomes indicate the
Single-cell RNA imaging localization of the integrated EBV in the genome.
Many prokaryotic and eukaryotic mRNAs are
targeted to specific regions of the cell. Cells are
highly compartmentalized. To image RNA in
single cells, the cells have to be fixed with
formaldehyde to preserve the cell shape
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller Jylan13. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $4.83. You're not tied to anything after your purchase.
