1. Analysis of Gene Expression - Overview -
Julia Paxson DVM DACVIM

2. Why gene expression analysis?
Quantification of mRNA transcript abundance
High specificity, +/- high through-put
Requires sequence knowledge

3. Considerations Experimental question Species limitations
Interpretation – post-translational modifications?
Technical limitations
Northern blot
Reverse transcription PCR
Quantitative PCR
Microarray analysis
RNAseq
Lower through-put
Cheaper
Less data analysis

4. Sample processing - mRNA degradation and induction
Store: RNAlater (Qiagen)
RNAprotect
TRIzol
Prep: TRIzol
Spin columns
QC: Electrophoresis
Nanodrop
Aligent Bioanalyzer

5. Northern blot analysis
Requires a large
sample size
Requires specifically
designed and labeled
RNA probes
Semi-quantitative
results

6. Polymerase chain reaction

7. Reverse transcription PCR
mRNA 
cDNA
PCR
Visualize
PCR product
using electrophoresis
Use florescence-labeled
probes/primers to follow
PCR in real-time
PCR product = exponential
NON-QUANTITATIVE
PCR product = linear
QUANTITATIVE

8. Quantitative PCR Uses fluorescence-tagged probes or primers
Relatively low through-put and single primer pair

9. Microarray analysis

10. DNA microarray Whole genome approach Relies on:
cDNA (amplification bias)
Hybridization
Species sequence info
Probe design

11. RNASeq
Aka deep sequencing, next-generation sequencing

12. RNASeq