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

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 Oct;7(10): How reliable is RNA-Seq data? Diermeier 2

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

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

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

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

One-Step vs Two-Step qRT-PCR Diermeier 7

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

How does qRT-PCR work? Diermeier 9

SYBR Green chemistry Diermeier 10

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 nt long Comparable GC content and T m (~60 °C) Length of amplicon: 60 – 200 bp Should span exon-exon junctions Diermeier 11

Primer Design: exon-exon junctions Diermeier 12

Primer Design: Databases Diermeier 13

Primer Design: Software Recommended software for primer design: Primer3 ( Primer-BLAST ( Benchling Geneious Diermeier 14

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

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

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

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

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

The 2 -ΔΔCT method Control gene Target gene Diermeier 20

The 2 -ΔΔCT method (Control, untreated) (Treatment) Diermeier 21

Essential: primer efficiency 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

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

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

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

The MIQE Guidelines Diermeier 26