Gene expression Flashcards
what can be used to detect mrna
northern blotting
in situ hybridisation
rt/qpcr
transcriptomics
reporter genes
what is northern blotting
its basically southern blotting for rna
sizes can be estimated by size markers
rna is denatured, formalydehyde included to strip secondary structure
ph is neutral or slightly acidic to preserve rna
fixed to membrane with uv
what does fish stand for
flourescent in situ hybridisation
what does fish involve
localisation of rna in samples
samples are fixed and dna removed
probes target specific sequences
what are q and rt pcr
quantatative pcr and reverse transcriptase
how does q and rt pcr funtion
first, cdna is created
then, sequence specific primers are bound
qt pcr counts pcr cycles
prescence of flourescent measuered by number of cycles needed to pass threshhold
what are the two forms of qpcr detection methods
non specific - uses intercalating dyes which flouresce under ds dna
requires primer specificity
specific - taqman molecular beacons
requires specific primers and probes
how do molecular beacons work
short probe has a reporter and a quencher, when hybridisation occurs, flourescent is moved away from quencher
how does taqman work
pcr amplification displaces and degrades taqman releasing flourescent
what are reporter genes
easily detectible proteins
they may generate a chromo or flouro phore, either directly or secondarily
what are some reporter gene examples
lacz
gfp
luciferase
cat
what are transcriptomics
the profiling of the entire rna of a cell
what are the two methods to measure gene expression
microarray
rna seq
how do microarrays function
thin sillycone wafers have genes printed onto them
cdna is produces and flourescently(red and green)
dna is added to plate, washed, and the color is measured. the resultant color shows how expression changes
how does rna seq function
fragment, cdna, sequence
align reads to each other, read depth relates to expression
what are the differences between microarrays and rna seq
microarrays can measure changes of known transcripts
dynamic range 44 fold
rna seq
higher dynamic range
can find new genes as well
what are the stages for analysing transcriptomic data
quality control
data preprocessing
differential gene analysis
clustering
network analysis
what is involved in quality control of transcription expression data
removing bad data sets such as outliers
removing bad reads
normalize data
what can normalisation do to data
it can distort biological differences, and llower the amount of genes you would think are related
what is a log transformation
it turns ratios into low log numbers that are easier to compare
what are some tests for comparing more than two conditions
limma
anova
how do you control for multiple comparisons
you use the bonferroni correction
a’ = a/k
new threshhold a’
old threshold a
number of conditions k
what is cluster analysis
the clustering of genes based on patterns of expression
distance can be used as a measure of similarity
what are gene ontologies
analysis of gene function to allow for and as a product of clustering
