Lecture 4: single-cell gene expression Flashcards
How is it that different cell types in our body contain the same DNA, but still are different from each other?
The DNA is the same, but gene expression in cells is different.
Some information about mRNA, just read.
- It is the molecule that transfers the information from the DNA to the protein.
- It participates in translating the genotype into a phenotype.
- It is a key determinant of protein abundance (not the only determinant).
- Changes in mRNA expression allow the cell to adapt to changing environments by responding to stimuli.
- Several mechanisms control mRNA levels, such as transcription, splicing, mRNA decay.
Why study mRNA expression instead of protein expression?
(Most) regulation of gene expression starts during RNA expression. RNAs don’t have to be translated into proteins to have a function in cells. So studying RNA expression is important to understand cell behaviour.
What is meant by the fact that mRNA expression is mostly studied by bulk analysis?
It means that multiple cells are used to study RNA expression.
What is the definition of low-to-mid-plex techniques of studying mRNA expression and what is the definition of higher-plex techniques? Also give two examples of these techniques.
- Low-to-mid-plex techniques are techniques used to study expression of 1-10 mRNAs. Example is Northern blot and RT-PCR or qRT-PCR.
- Higher-plex techniques are used to study expression of >10 mRNAs. Example is DNA microarray or RNA-sequencing.
What is Northern blotting?
The size and the amount of RNA can be studied through Northern blotting. The steps are:
- Cells from tissue are collected and cleaved.
- RNA is isolated and seperated based on size by electrophoresis.
- The separated RNA is transfered on a membrane
- Probes with a fluorescent tag are used that can hybridize/bind to RNA of interest
- Bands are visualized and size and intensity/amount can be measured.
What is RT-PCR and qRT-PCR?
(q)RT-PCR stands for (quantitative) Reverse Transcriptase-Polymerase Chain Reaction. Here, the starting point is (isolated) RNA from extracted cells. Reverse transcriptase is used to synthesize double stranded cDNA. The cDNA is amplificated by PCR with the use of fluorophores/intercalating dyes. These fluorophores emit fluorescent light that can be used to quantify the amount of double stranded cDNA.
What is DNA microarray?
- A chip is used with DNA-probes bound to the chip (resembeling (part of) a genome).
- Then cells of (e.g.) healthy and tumor tissue is extracted and mRNA is isolated.
- cDNA is synthesized from the isolated mRNA with the use of reverse transcriptase and are fluorescently labeled. Here, e.g. the healthy tissue is labeled with GFP while the tumor tissue is labeled with RFP.
- The cDNA is then added to the chip and hybridizes with their complementary DNA-probes.
This way it can be researched what genes are expressed in what kind of tissue.
RNA sequencing (specifically Illumina sequencing) consists of four steps:
- Sample preparation
- Bridge amplification
- Parallel sequencing
- Data analysis
Explain step 1
mRNA is isolated and added to a flow cell containing poly(T) sequence primers (oligo) that bind to mRNA poly(A) tails. When mRNA binds to these primers, a double stranded cDNA is made. The dsDNA is denatured and original mRNA is washed away.
RNA sequencing (specifically Illumina sequencing) consists of four steps:
- Sample preparation
- Bridge amplification
- Parallel sequencing
- Data analysis
Explain step 2
The single stranded DNA still attached to the flow cell binds to another type of oligo/primer on the flow cell, where polymerases generate the complementary strand. This bridge is denatured, forming two single stranded copies. This process is then repeated. At last, the reverse strands are washed off.
RNA sequencing (specifically Illumina sequencing) consists of four steps:
- Sample preparation
- Bridge amplification
- Parallel sequencing
- Data analysis
Explain step 3 and 4
A sequencing primer starts of the process, whereafter complementary nucleotides one by one bind to the forward strand. The nucleotides are fluorescently tagged, which a computer picks up (sequencing by synthesis).
RNA sequencing (specifically Illumina sequencing) consists of four steps:
- Sample preparation
- Bridge amplification
- Parallel sequencing
- Data analysis
How is all the mRNA finally sequenced so that e.g. genetic variety can be identified?
The idea is that through the steps in Illumina sequencing and the use of various primers, different lengths of reverse and forward strand are made of the mRNA that is researched. Together these form a contig (a set of overlapping DNA segments that together represent a consensus region of DNA.
What kind of information is missed by techniques like RT-PCR, microarray or RNA sequencing?
- Cell-to-cell heterogeneity → one isolated cell doesn’t represent all the other cells (even the same types).
- Spatial information, where the mRNA is made (sub-cellular, within tissue/organism).
Name three reasons why it is important to study gene expression in single cells.
- Researching more cells and thus usually only being able to measure averages, will not show the cells of interest if they’re in the minority (picture).
- Cells with the same genome do not express the same mRNAs and proteins
- To identify why cells become drug-tolerant to e.g. cancer treatments or antibiotics.
Think of disadvantages and advantages for these techniques.
How do you perform mRNA imaging to study mRNA distribution and what is this technique called?
In situ hybridization
- Fix cell with formaldehyde (with this preserving cell shape and mRNAs)
- In situ hybridization with isotopically labelled probes
- Detection by exposure to photographic emulsion (picture)
How do ZIP-codes control mRNA localization?
These cis-acting motifs are found in the untranslated mRNA region (UTR) or in the coding sequence. They can form 3D structures that are recognized by RNA binding proteins and molecular motors.
What can be seen in this picture?
Localized translation of β-actin in mammalian fibroblasts.
There are four methods available to visualize mRNAs in living cells:
- RBP/RNA tag (MS2/PP7 system)
- Fluorogenic RNA
- Molecular beacons
- rCas9/dCas13
Briefly explain these methods.
- RBP/RNA tag (MS2/PP7 system) → the tag is composed of a RNA binding protein (RBP) that is bound to a fluorophore. It can then bind to mRNA and fluorescence can be measured/visualized.
- Fluorogenic RNA → modify mRNA with RNA aptamer. The RNA aptamer will only emit light when its ligand binds.
- Molecular beacons → this beacon consists of a DNA hairpin structure containing a quencher and fluorophore. The fluorophore doesn’t emit any light if its close to the quencher. Only when the hairpin structures is denatured and the DNA binds to its target sequence, the quencher and fluorophore are seperated and the fluorophore will emit light.
- rCas9/dCas13 → the CRISPR-Cas system is used, where a Cas13 protein is tagged with GFP and guide RNA is used that guides Cas13 to its target RNA.
The MS2 tagging system is based on the coat protein of the MS2 bacteriophage. Why?
This coat protein contains an RNA-binding site with high binding affinity for RNA stem-loop/hairpin structures found only in the bacteriophage RNA. To identify and localize mRNA in living cells, two plasmids are used: one plasmid transcribes the transcript of interest bearing multiple MS2 hairpins and one plasmid containing the MS2 binding/coat protein with GFP tagged. This way the RNA of interest is recognized by the MS2 coat protein via the MS2 hairpin structures attached to the RNA of interest.
mRNA can also be measured to study export dynamics of mRNA. How was this done and what conclusion is there?
The nuclear envelope was visualized and beta-actin mRNA (just an example) was visualized. This will tell you how long mRNA stays inside the nucleus or in the cytoplasm. They saw that mRNA spends the shortest time around the nuclear envelope. See picture.
How is beta-actin mRNA localization visualized in the neurons of mice?
With the use of the MS2 system in mice, where one mouse with GFP+coat protein MS2 is crossbred with a mouse with beta-actin mRNA and hairpins.
What results were found when beta-actin mRNA was localized in neurons?
mRNA is moving around dendrites and when neurons are stimulated, mRNA localizes and is (usually) translated there into a protein.
