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
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
What is transcriptomics?
“Study of transcriptome, the complete set of RNA transcripts produced by a give cell population or tissue under specified conditions”
What are the two main methods of measuring the transcriptome
- Microarrays
2. Next-generation sequencing
How can we study all genes simultaneously
- Spotted microarrays
2. In situ synthesis
What are Affymetrix GeneChips
- 25-mer oligos synthesised directly on
chip - photolithographic technology
- up to 5,300,000 oligos per chip
- Each gene represented by many oligos,
ensures that some bind efficiently
how and why is RNA converted to cDNA and labelled
- RNA is converted to complementary DNA
(cDNA) before adding to microarray
(mRNA -> cDNA = reverse transcription) - This is because cDNA is more stable,
and stability is needed - Fluorescent tags can be added to cDNA
(used to visualise the sports on arrays) - Comparing RNA from normal cells vs
cancer cells for eg., different coloured
tags can be used
How do you get information out of a microarray?
- once sample RNA is made and within
a cell, labelled targets can hybridise to
form cDNA of set colour
What are the pros and cons of microarrays
+ monitor gene expression of all genes
in an organism at a given time or
condition
+ Relatively inexpensive
+ Relatively fast (in relation to amount of
data produced)
- Need a genome
- V sensitive to environmental changes
- Need to validate biological significance
- Can be difficult to get enough good
quality RNA from some samples
What is “next-generation sequencing of the transcriptome”?
1. extract RNA from samples of interest.
E.g:
a. healthy tissue
b. tumour
2. (Generate cDNA), fragment, size
select, add linkers
3. sequencing (100s of millions of reads,
10s of billions bases of sequence)
4. Bioinformatics analysis:
a. Map reads to genome data set(s)
b. Generate information on gene
expression (transcriptomics) based
on number of reads
5. Many diff methods for sequencing
the transcriptome:
a. paired-end sequencing
b. single-read sequencing
c. RNA-seq
d. these give different data (e.g., read
length and depth)
6. Some methods can sequence RNA
directly (direct RNA sequencing),
other rely on RNA conversion to
cDNA as per arrays
7. Sequencing can give data on genes,
intergenic regions, splicing, and small
non-coding RNAsWhat are the pros and cons of Next-gen sequencing
\+ sequences everything, not just gene information (e.g., sRNA) \+ Don't necessarily need to know what your looking for \+ Don't need a genome sequence
- Cost still relatively high relative to
microarrays - Needs a lot of bioinformatics analysis to
interpret data - Time consuming
What is meant by “hidden deficiency”
“The wrong mic of gut bacteria, not just starvation, could contribute to severe malnutrition”
In Malawi, 10s of thousands of children have this, with 15% being fatal
