Gene Expression Flashcards
Why measure gene expression?
To understand differences between cells/tissues
To understand changes in genes during cancer etc
To better understand disease mechanisms better, to develop marker for disease - biomarkers
Methods to determine gene expression
1.expression reporters - visible protein products hitched to gene products
Microarray - expression level of many (all) genes simultaneously
qRT-PCR - confirmation of expression levels of selected genes
RNA seq - microarray level of expression depth but also info on mutations and splicing
1.expression reporter genes - promoter/enhancer sequence is attached to gene whose product can be easily assayed
Lacz- beta galactosidase
GFP - green fluorescent protein
Can use for whole organisms
Yep
2.northern blotting
Purify RNA from cells
Electrophoresis of RNA through gel
Transfer RNA to solid support - nitrocellulose membrane
Hybridise radioactive probe designed against target gene and one against control gene
Approximately equal to amount of RNA
3.microarrays
DNA attached to solid support(chip)
Each spot is a different DNA sequence (gene)
Sample RNA is fluorescently labelled
Hybridised to chip
Signal detection
Can be comparative (ratio between samples) or intensity based
Samples- mRNA- cDNA- DNA microarray (two channel)
Use of microarrays in expression profiling
Metabolic changes, drug induced expression changes, tumour States
Transgenic animal profiling
QC and analysis
Analysis of microarrays
Use a fluorescent scanner to quantitate the ratio/amount of probes bound to each spot
Need
1.quality control - removes very low expression or highly variable gene signals
Log base 2 of expression level compresses scale
Results often checked using qRT-PCR
Typical microarray scatter plots
Eg genes affected by drug treatment. Can be down regulated, upregulated
Often described as fold change decrease / increase
Volcano plots tell us about both fold change and robustness = variance in samples
Microarray cluster analysis can place genes in groups by similarity - personalised medicine and biomarkers
4 quantitative PCR based methods of gene expression analysis
First need to reverse transcribe cDNA Then normal PCR of cDNA Called RT-PCR Hybridise with oligo DT primer Transcribe RNA to cDNA Quantisation reveal doubling each round of annealing extension
To measure the RTpcr after each cycle
Run on a gel after each cycle, stain with a dye (sybr green) that binds dsdna (PCR products) can see increase in tube
Can’t bind single or primer
Amount of fluorescence is proportional to amount of dsDNA
Dynamic and tightly regulated gene expression
Circadian gene expression
Immediate early genes, ID-miRs, DEGs (delayed), slow response genes
Summary on gene expression
mRNA levels can be measured in a variety of ways:
Reporter assays
Northern blots
QRTpcr
Microarrays
Rnaseq
