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Analysis of Gene Expression - Overview -

Analysis of Gene Expression - Overview -. Julia Paxson DVM DACVIM. Why gene expression analysis?. Quantification of mRNA transcript abundance High specificity, +/- high through-put Requires sequence knowledge. Considerations. Experimental question Species limitations

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Analysis of Gene Expression - Overview -

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  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 AligentBioanalyzer

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

  6. Polymerase chain reaction

  7. Reverse transcription PCR mRNA  cDNA  PCR Visualize PCR product using electrophoresis Use florescence-labeled probes/primers to follow PCRin 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

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