Lecture 25: Functional Genomics 1 Flashcards
Why does it matter to know when and where a gene is expressed?
- Gene expression can be v specific:
a. Cell and/or tissue types
b. Times during development
c. Under particular conditions (e.g., stress)
d. During disease (e.g., cancer) - Understanding when and where can be
important for understanding gene
function
What are different techniques used to determine when and where a gene is expressed?
- In situ hybridization
- Promoter-reporter studies
- Northern blotting
- Quantitative real-time PCR (qRT-PCR)
What is In situ hybridization?
“In vivo technique to detect specific RNA sequences”
- Suitable sample taken from a culture and
transferred to slide - Labelled probe (DNA or RNA) is added
to the slide causing “hybridization of
labelled probe to target RNA” - Unbound probe gets washed away so
bound probe can be visualised
What are the advantages and disadvantages of in situ hybridization?
\+ no RNA extractions required \+ Gives spatial info about cell and tissue here gene is expressed \+ Different labels can be used, e.g., chromogenic or fluorescent labels
- Can be technically challenging to set-
up and time consuming (one gene at a
time)
What are promoter-reporter studies?
“in vivo technique to detect expression driven by specific gene promoters”
Different reporter genes:
- Green fluorescent protein (GFP)
- Beta-galactosidase (beta-Gal)
- Glucuronidase (GUS)
What are the advantages and disadvantages of promoter-reporter studies
+ No RNA extractions required
+ Low cost
+ Gives spatial info about cells/tissues
where gene is expressed
- Need to be able to genetically
manipulate your species of interest - Need to define a promoter region
which is functional
What is meant by RNA extraction
1. Various RNA extraction methods
available as a "kit"
2. RNA is extracted from a given sample:
a. bacterial cultures
b. plant cells
c. animal cells
3. Different tissues/cells can be selected.
e.g;
a. traditional dissection
b. laser capture microdissection
c. fluorescence activated cell sortingHow does RNA extraction work?
- RNA stabilization from cell
- Cell lysis & homogenisation
- Protein precipitation
- DNase treatment
- RNA purification
What is meant be “RNA quality”
- mRNA can be very unstable
- Most important factor for successful
analysis of gene expression - Assess on gels
- Good ribosomal RNA bands are
reassuring
What is northern blotting?
“in vitro technique to detect specific RNA sequences”
- RNA extraction from sample
- Electrophoresis of RNA (RNA separated
by size) - Northern Blotting (transfer RNA to
membrane) - Label RNA with radioactively labelled
probes - Visualise labelled RNA on X-ray film
What are the pros and cons of Northern Blotting?
+ Method is relatively simple and low cost
+ mRNA transcript size can be determined
+ Alternatively spliced RNA transcripts
can be detected
- One gen at a time
- Difficult to quantify amount of transcript
- Not as sensitive as other techniques
(e. g., qRT-PCR) - Need to use radioactively
What is qRT-PCR
“in vitro technique to quantify mRNA and cDNA”
- RNA extraction from sample
- Reverse transcription of mRNA to cDNA
- PCR using fluorescent dyes used to
quantify amount of DNA amplified in each
PCR cycle
What are the pros and cons of qRT-PCR
+ highly sensitive
+ Quantitative method
- Relatively expensive vs traditional PCR
(special reagent and PCR machine
required - One gene at a time (low throughput)
How can we predict genes of unknown function
Bioinformatics analyses (e.g., “gene ontology”)
What is functional genomics
“Field of molecular biology attempting to make use of the vast wealth of data produced by genomic projects”
Unlike genomics, functional genomics focusses on dynamic aspects such as gene transcription & translation, and protein-protein interactions
TRIES TO IDENTIFY GENE AND PROTEIN FUNCTIONS
