Expression Profiling Flashcards
Define transcriptiome.
The set of all mRNA molecules in one cell or a population of cells.
When observing the transcriptiome, how can the expression of a gene indicative of function be analysed?
By the site of expression and the size/content of the transcripts.
What are the three methods of single gene analysis?
- Northern blotting.
- RT-PCR.
- In situ hybridisation.
What are the three methods of wider analysis of the transcriptiome?
- Expressed sequence tags.
- Microarrays.
- Sequencing.
State the purpose of Northern blotting.
Studying gene expression by detection of RNA (or isolated mRNA) in a sample.
In Northern blotting, how are RNA samples denatured?
At 90°C and using formamide.
In Northern blotting, how are RNA samples separated?
By size electrophoresis in a denaturing agarose gel.
In Northern blotting, what type of denaturing agarose gel is used?
Formaldehyde.
In Norther blotting, what happens to the RNA after separation?
It is transferred to a nylon membrane, crosslinked and then hybridised with a labelled probe.
In Northern blotting, how is mature mRNA isolated?
Only those with a poly(A) tail are isolated.
In Northern blotting, what are the three types of hybridisation probes?
- Radiolabelled/non–radiolabelled labelled DNA.
- In vitro transcribed RNA.
- Oligo-nucleotides.
In Northern blotting, what is the alternative probe to hybridisation probes?
Sequences with partial homology.
What four types of information can Northern blotting deduce?
- Sites of expression.
- Level of expression.
- Size of transcript.
- Alternative spliceforms.
State the purpose of RT-PCR.
Amplify specific mRNAs by means of gene specific primers.
What three pieces of information does RT-PCR provide?
- Sites of expression.
- Level of expression (semi-quantitative).
- Alternative spliceforms.
RT-PCR qualitatively detects gene expression through creation of what?
Complementary DNA (cDNA) transcripts from RNA templates.
In PT-PCR, what are the three types of primers?
- Random.
- Oligo (dT).
- Sequence specific.
In PT-PCR, how is the locus amplified?
By means of specific primers.
In digital RT-PCR, why is the sample partitioned?
So individual RNA molecules within the sample are loacalised within various separate regions.
In digital RT-PCR, what type of distribution of molecules do you assume?
Poisson.
In digital RT-PCR, what does 0 represent?
A negative result.
In digital RT-PCR, what does 0 represent?
A positive result.
What is the last step of digital RT-PCR?
Absolute quantitation of number of copies of target.
How are tissues prepared for in Situ hybridisation?
They are cut by a microtome and then either frozen or embedded in wax.
During in Situ hybridisation, why is hybridisation of a suitable gene-specific probe to the tissue use?
To provide detailed expression images representative of the distribution of the RNA in the tissue.
What is the purpose of whole-mount in situ hybridisation?
To hybridise intact tissues or embryos.
In Whole-mount in situ hybridisation, what are the probes labelled with?
UTP-digoxigenin.
In Whole-mount in situ hybridisation, what affects the permeability of the cells in order to open the cell membrane?
Proteinase K.
In Whole-mount in situ hybridisation, the labelled probe binds to what substance?
mRNA.
In Whole-mount in situ hybridisation, what happens to the cell after the probe has been bound?
It is washed.
In Whole-mount in situ hybridisation, what type of antibody is used?
Alkaline phosphate-conjugated antibody.
In Whole-mount in situ hybridisation, what does the antibody bind to?
Digoxigenin.
In Whole-mount in situ hybridisation, how does the dye process work?
A colourless compound is added to the cell, which become purple when phosphate is removed.
Define differential display.
Identifying changes in expression in mRNA transcripts between two cell samples.
What two methods does differential display entail?
Partially degenerate PCR primers and modified RT-PCR for studying the expression of numerous genes simultaneously.
State an advantage of using differential display.
There is no need for previous information about expressed genes.
Define ESTs.
Small fragments of DNA sequence which is generated by sequencing either one or both ends of an expressed gene.
How many nucleotides long are ESTs?
200 to 500.
What are two advantages of ESTs?
Random and unbiased.
ESTs can reconstruct complete sequences of what?
cDNA.
What four pieces of information can be derived from ESTs?
- The cells where the gene is expressed.
- Level of transcripts (semi-quantitative).
- Isoforms (alternate promoter usage and alternative splicing).
- Homologs in other species.
What does dbEST (Genbank) stand for?
Database of EST sequences.
What is the purpose of Unigene within Genbank?
It automatically separates sequences into a non-redundant set of gene-oriented clusters.
What are the two types of DNA chips?
cDNA (spotted by robot) and oligonucleotide (in situ synthesis by photolithography).
State the four steps of microarrays.
- Isolate mRNA from tissue.
- Convert mRNA to first-strand cDNA.
- Degrade mRNA with NaOH and label cDNA.
- Hybridise fluorescent labelled cDNA with microarray chip and detect fluorescence.
In regards to microarrays, does a human or a robot spot the genes onto a slide?
Robot.
In regards to microarrays, why is the mRNA collected from two different cell samples compared?
For a direct comparison of their relative levels of gene expression.
In regards to microarrays, what is the mRNA samples converted into?
cDNA.
In regards to microarrays, cDNA from one source (control) is labelled with Cy3? What colour does this present as?
Green.
In regards to microarrays, cDNA test is labelled with Cy5. What colour does this present as?
Red.
In regards to microarrays, what happens to the labelled samples?
They are mixed in order to hybridise to become a microarray.
In regards to microarrays, after incubation is complete, what occurs to the array?
It is washed and fluorescently scanned.
In regards to microarrays, what does a green spot indicate?
The expression of the gene is higher in the control sample.
In regards to microarrays, what does a red spot indicate?
The gene in the test sample is expressed at a higher level than the corresponding gene in the control sample.
In regards to microarrays, what does a yellow spot indicate?
Genes are expressed at equal levels in both samples.
In regards to microarrays, what does a dark spot indicate?
Little or no expression in either sample of the gene whose fragment is located at that position in the array.
In regards to microarrays, what alternative manner can data be represented as?
A heatmap.
Explain how a heatmap works in regards to the grid layout (rows and columns).
Each row represents a different gene and each column corresponds to different samples.
In regards to a heatmap, areas that are predominately green represent what?
Down regulated genes in multiple samples.
In regards to a heatmap, areas that are predominately red represent what?
Up regulated genes in multiple samples.
In regards to a heatmap, areas that are black represent what?
Genes whose expression is not changed between samples.
In regards to a heatmap, shade variations demonstrate what?
The degree of up or down regulation between samples.
Define cluster analysis in regards to genetics.
Identify sets of genes that are coordinately regulated.
State three advantages of sequencing the transcriptiome.
- Development of next generation sequencing technologies.
- Enable high throughput sequencing of multiple molecules.
- Unbiased.
State four functions of single cell RNA-sequencing.
- Isolate single cells.
- Isolate mRNA.
- cDNA synthesis.
- RNA sequence.
Name four methods to isolate single cells.
- Fluorescence-activated cell sorting.
- Optofluid-based cell handling.
- Microfluidic-based cell handling.
- Laser-capture microdissection.
State three advantages of single cell RNA-sequencing.
- No issue with heterogeneity in cell types from tissue.
- Individual cancer cells from a tumour (no contaminating cells).
- Inter-cell variation in transcriptiome.
