Overview Why gene expression analysis Quantification of mRNA transcript abundance High specificity high throughput Requires sequence knowledge Considerations Experimental question Species limitations ID: 917582
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Slide1
Julia Paxson DVM DACVIM
Analysis of Gene Expression
- Overview -
Slide2Why gene expression analysis?
Quantification of mRNA transcript abundance
High specificity, +/- high through-put
Requires sequence knowledge
Slide3Considerations
Experimental question
Species limitations
Interpretation – post-translational modifications?Technical limitations
Northern blotReverse transcription PCRQuantitative PCRMicroarray analysis
RNAseq
Lower through-put
Cheaper
Less data analysis
Slide4S
ample processing - mRNA degradation and induction
Store:
RNAlater
(
Qiagen
)
RNAprotect
TRIzol Prep: TRIzol
Spin columns
QC: Electrophoresis
Nanodrop
Aligent
Bioanalyzer
Slide5Northern blot analysis
Requires specifically
d
esigned and labeled
RNA probes
Requires a large
sample size
Semi-quantitative
results
Slide6Polymerase chain reaction
Slide7Reverse transcription PCR
mRNA
cDNA
PCRVisualizePCR productusing electrophoresis
Use florescence-labeled
probes/primers to follow
PCR
in real-time
PCR product = exponential
NON-QUANTITATIVE
PCR product = linear
QUANTITATIVE
Slide8Quantitative PCR
Uses fluorescence-tagged probes or primers
Relatively low through-put and single primer pair
Slide9Microarray analysis
Slide10DNA microarray
Whole genome approach
Relies on:
cDNA (amplification bias)Hybridization Species sequence info
Probe design
Slide11RNASeq
Aka deep sequencing, next-generation sequencing
Slide12RNASeq
Slide13