1. Validation of RNA-Seq data An introduction to qPCR Sarah Diermeier, Ph.D. Cold Spring Harbor Laboratory 4-21-2016

2. Are differential expression changes accurate? Are novel transcript isoforms real? See ALEXA-Seq publication for details: Griffith et al. Alternative expression analysis by RNA sequencing. Nature Methods. 2010 Oct;7(10):843-847. How reliable is RNA-Seq data? Diermeier 2

3. Qualitative RT–PCR Sanger sequencing (SNPs, new exon-exon junctions) in situ hybridization (RNA-FISH) Validation methods for RNA-Seq data Diermeier 3

4. RNA-FISH: Validation and Localization Zhang et al., Cell Reports 2012 Diermeier 4

5. Qualitative RT–PCR Sanger sequencing (SNPs, new exon-exon junctions) in situ hybridization (RNA-FISH) Quantitative Northern Blotting Microarray quantitative Real Time – PCR (qRT-PCR) Validation methods for RNA-Seq data Diermeier 5

6. Griffith et al., Nature Methods 2010 RNA-Seq and qRT-PCR data correlate well Diermeier 6

7. One-Step vs Two-Step qRT-PCR http://www.thermoscientificbio.com Diermeier 7

8. How does qRT-PCR work? VanGuilder et al., BioTechniques 2008 Diermeier 8

9. How does qRT-PCR work? www.affymetrix.com Diermeier 9

10. SYBR Green chemistry http://www.thermoscientificbio.com Diermeier 10

11. Primer Design: General Guidelines Criteria for the design of qRT-PCR primers: Unique for the target sequence Low probability for primer-dimer and secondary structure formation 18-25 nt long Comparable GC content and T m (~60 °C) Length of amplicon: 60 – 200 bp Should span exon-exon junctions Diermeier 11

12. Primer Design: exon-exon junctions http://www.thermoscientificbio.com Diermeier 12

13. Primer Design: Databases Diermeier 13

14. Primer Design: Software Recommended software for primer design: Primer3 (http://bioinfo.ut.ee/primer3/) Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast) Benchling Geneious Diermeier 14

15. Primer Design: Specificity Verify primer specificity: Primer-BLAST Optimize T m Melting Curve Electrophoresis of the qRT-PCR product Multiple primer pairs per target Diermeier 15

16. Example: Vsx1 expression in mouse Watson et al., Molecular Vision 2011 Diermeier 16

17. Example: Vsx1 expression in mouse Watson et al., Molecular Vision 2011 Diermeier 17

18. Melting Curves validate specificity Robert et al., Biology of Reproduction 2002 Diermeier 18

19. Quantitation using the 2 -ΔΔCT method VanGuilder et al., BioTechniques 2008 Diermeier 19

20. The 2 -ΔΔCT method http://www.appliedbiosystems.com Control gene Target gene Diermeier 20

21. The 2 -ΔΔCT method http://www.appliedbiosystems.com (Control, untreated) (Treatment) Diermeier 21

22. Essential: primer efficiency http://www.appliedbiosystems.com E = 10^(-1/slope) Requirement for the 2 -ΔΔCT method: Efficiencies of the target gene and the control gene amplification must be similar Diermeier 22

23. A note about control genes VanGuilder et al., BioTechniques 2008 Diermeier 23

24. Endogenous control genes “Housekeeping“ genes Expression levels must be Similar in all tested samples Resistant to experimental conditions Following the same qRT-PCR kinetics as the target genes (primer efficiency) Diermeier 24

25. Endogenous control genes Commonly used examples: GAPDH β-actin Tubulin 18S or 28S rRNA Ribosomal proteins More resistant and accurate: geometric mean of three control genes (“basket” normalization) Diermeier 25

26. The MIQE Guidelines Diermeier 26