Successful gene expression studies using validated qpcr assays. Jan Hellemans, CEO Biogazelle webinar October 28 th, 2015
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1 Successful gene expression studies using validated qpcr assays Jan Hellemans, CEO Biogazelle webinar October 28 th, 2015
2 Agenda Requirements for high quality qpcr assays Approaches for qpcr assay validation How good a result can be achieved with highly optimized design tools How to easily discover additional genes of interest Best practice in qpcr
3 Online poll How would you rank your current knowledge about gene expression assay design? a) basic b) advanced c) expert
4 Introduction qpcr is reference technology for nucleic acid quantification sensitivity and specificity wide dynamic range speed relatively low cost conceptual and practical simplicity qpcr is easy to perform easy to do it right many steps involved all need to be right
5 Prepare cycle report experiment design samples assays P C R prepare cycle report relative quantification quality control statistical analysis
6 Prepare cycle report experiment design samples assays P C R prepare cycle report relative quantification quality control statistical analysis
7 Assay design & validation Considerations & best practices design amplicon length primer positions (exonic or intron-spanning)
8 Assay design & validation Considerations & best practices design amplicon length primer positions (exonic or intron-spanning) gene exonic intron-spanning
9 Assay design & validation Considerations & best practices design amplicon length primer positions (exonic or intron-spanning) transcript coverage gene transcript 1 transcript 2 transcript 3 coverage
10 Assay design & validation Considerations & best practices design amplicon length primer positions (exonic or intron-spanning) transcript coverage in silico verification specificity prediction (retropseudogenes and other homologues) secondary structure analysis wet lab validation (experimental) specificity assessment (gel, melt, amplicon sequencing) Cq of NTC (for SYBR assays) amplification efficiency determination (slope, E, SE(E), r2)
11 Assay design & validation Considerations & best practices design amplicon length primer positions (exonic or intron-spanning) transcript coverage in silico verification specificity prediction (retropseudogenes and other homologues) secondary structure analysis wet lab validation (experimental) specificity assessment (gel, melt, amplicon sequencing) Cq of NTC (for SYBR assays) amplification efficiency determination (slope, E, SE(E), r2)
12 Properties of the perfect assay specific for the gene of interest => no off-target amplification detection of all transcript variants detection not affected by polymorphisms => no allelic bias or drop out amplification efficiency ~100% no gdna co-amplification no primer dimer formation
13 The perfect assay
14 Online poll How do you currently obtain your perfect qpcr assay? a) using your own home brewed assays b) buying pre-designed assays (commercial) c) currently not designing any gene expression assays
15 The perfect assay... or the best possible For some genes, there is no perfect assay no unique sequence (homology with other genes pseudogenes)
16 Gene homology in olfactory receptor genes prevents perfect designs distances (clustalw) between all genes without perfect design 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% / 2043 (75%) of genes without perfect design have homologous genes that differ less than 12.5% (2 variations per 16 bases)
17 The perfect assay... or the best possible For some genes, there is no perfect assay no unique sequence (homology with other genes pseudogenes) no common sequence among all transcripts regions are excluded because of repeats, secondary structures, SNPs, homology,... Make the best possible compromise and report potential issues Design in silico quality control wet lab validation
18 Assay design using primerxl database of genomic information (transcripts, SNPs,...) tools for target region selection (maximize transcript coverage) primer3 design engine analysis of secondary structures and SNPs in primer & probe annealing regions specificity prediction (BiSearch, bowtie) relaxation cascade (from perfect to best possible)
19 Impact of primer mismatches on qpcr assay performance Lefever, Clin Chem 2013
20 BiSearch specificity prediction BiSearch loose BiSearch strict
21 BiSearch specificity prediction BiSearch loose BiSearch strict only the gene of interest (FFAR2) reads seq gene_list official_symbol location 2843 CATGGCAGTCACCATCTTCTGCTACTGGCGTTTTGTGTGGATCATGCTCTCCCAGCCC CTTGTGGGGGCCCAGAGGCGGCGCCGAGCCGTGGGGCTGGCTGTGGTGACGC TGCTCAATTTCCTGGTGTGCTTCGGACCTTACAGATCGGAA 1897 GTAAGGTCCGAAGCACACCAGGAAATTGAGCAGCGTCACCACAGCCAGCCCC ACGGCTCGGCGCCGCCTCTGGGCCCCCACAAGGGGCTGGGAGAGCATGATCC ACACAAAACGCCAGTAGCAGAAGATGGTGACTGCCATGAGATCGGAA 1535 GTAAGGTCCGAAGCACACCGAGAGCTGGGAGCAGGAGCTACACAGTCTGCTGG CCTCACTGCACACCCTGCTGGGGGCCCTGTACGAGGGAGCAGAGACTGCTCCT GTGCAGAATGAAGGCCCTGGGGTGGAGATGCTGCTGTCCTCAGAA 1097 CATGGCAGTCACCATCTTCTGAGGACAGCAGCATCTCCACCCCAGGGCCTTCATT CTGCACAGGAGCAGTCTCTGCTCCCTCGTACAGGGCCCCCAGCAGGGTGTGCA GTGAGGCCAGCAGACTGTGTAGCTCCTGCTCCCAGCTCTCGG 1091 CATGGCAGTCACCATCTTCTGAGGACAGCAGCATCTCCACCCCAGGGCCTTCATT CTGCACAGGAGCAGTCTCTGCTCCCTCGTACAGGGCCCCCAGCAGGGTGTGCA GTGAGGCCAGCAGACTGTGTAGCTCCTGCTCCCAGCTCTCGGT ENSG FFAR2 19: ENSG FFAR2 19: ENSG AC : ENSG AC : ENSG AC :
22 Wet lab validation setup PCR composition total volume: 5 μl instrument: Bio-Rad CFX384 (with CFX Automation System) mastermix: Bio-Rad SsoAdvanced SYBR primer conc: 250 nm each PCR program default cycling protocol for SsoAdvanced SYBR (Ta=60 C) Samples cdna: 25 ng (total RNA equivalents Agilent Universal human reference RNA = MAQC A) gdna: 2.5 ng (Roche) NTC: water + carrier (5 ng/μl yeast transfer RNA) synthetic template (pooled 60-mers in concentration range: 20 M 20 copies)
23 Wet lab validation some numbers 305 m lab validation of assays (human, mouse and rat coding genes) reactions PCR plates (384-well) equivalent to PCR plates (96-well)
24 Amplification efficiency synthetic templates initial publication: Vermeulen et al., Nucleic Acids Research, 2009 Biogazelle approach (easy & cost effective) 60-mer 30 nt 5 no modifications, standard desalted 30 nt 3 7 points dilution series: > 20 molecules equivalent to full length double stranded template ds template ss oligo r2< median E average E count E <> [ ] 1 3 paired t-test p-value 0.14 limitation: behavior of first cycles amplifying from cdna are not evaluated
25 Amplification efficiency distribution (n = ) 89%
26 Amplification efficiency distribution (n = ) redesign 89% redesign
27 Specificity NGS for increased sensitivity amplicon sizing ( + melt analysis for SYBR assays) limited sensitivity for detecting low level non-specific coamplification failure to observe non-specific amplification of sequences with similar size and/or Tm e.g. expressed pseudogenes or homologous genes next level of specificity assessment in silico specificity predictions by BiSearch massively parallel sequencing of pooled PCR products average coverage > 1000-fold lab specificity > 99.9% times more sensitive than size analysis and Sanger sequencing
28 Specificity most assays are 100% on-target
29 Specificity 2/3 of non-specific assays may go unnoticed without NGS % on-target 100% 75% 50% 25% 0% 0.9 < x < < x < < x < < x < < x < < x < < x < < x < < x < < x < 0.1 0% 20% 40% 60%
30 Specificity the power of in silico verification perfect % acceptable (<10% non-specific) % predicted non-specificity (no specific design found) % failing specificity QC criteria %
31 Online poll How do you validate your assay s specificity? a) Melt curves b) Size analysis (gel or capillary) c) Restriction digestion with gel analysis d) Sequencing of PCR products
32 Online poll Do you know the MIQE initiative? a) Yes b) No
33 MIQE compliant PrimePCR assay validation data sheet for human, mouse & rat
34 PrimePCR assay for 9 extra organisms building on the confidence validated on > 100,000 assays skip wet lab validation 9 organisms 27,155 19,762 19,310 25,006 15,307 20,184 19,049 6,572 21,360 assays for SYBR or with probe
35 Screening vs targeted Transcriptome microarray or RNA-seq hypothesis generating few samples (high cost per sample) Gene panels qpcr (high sensitivity, low cost) selection of genes based on pathway, cell type or disease convenient balance between blind screening and targeted analysis Individual gene(s) qpcr high flexibility, customization low cost per sample
36 Saturation analysis MAQC A - % of PrimePCR 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% ribo-depletion RNAseq - detection poly-a RNAseq - detection qpcr - detection ribo-depletion RNAseq - quantification poly-a RNAseq - quantification qpcr - quantification 0%
37 Questions?
38 Thank you for your attention!
Insights from the first RT-qPCR based human transcriptome profiling based on wet lab validated assays
Slide 1 of 38 Insights from the first RT-qPCR based human transcriptome profiling based on wet lab validated assays Jan Hellemans, PhD CEO Biogazelle qpcr & NGS 2013 Freising, Germany March 19, 2013 Biogazelle
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Gene Information Gene Name keratin 78 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID KRT78 Human This gene is a member of the type II keratin gene family
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Gene Information Gene Name collagen, type IV, alpha 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID COL4A1 Human This gene encodes the major type IV alpha
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Gene Information Gene Name major histocompatibility complex, class II, DR beta 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID HLA-DRB1 Human HLA-DRB1 belongs
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID mcf.2 transforming sequence-like Mcf2l Mouse Description Not Available C130040G20Rik,
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID Glyceraldehyde-3-phosphate dehydrogenase Gapdh Rat This gene encodes a member of
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)
PrimePCR Assay Validation Report
Gene Information Gene Name integrin, alpha 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID ITGA1 Human This gene encodes the alpha 1 subunit of integrin
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID glyceraldehyde-3-phosphate dehydrogenase GAPDH Human The product of this gene catalyzes
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID caspase 9, apoptosis-related cysteine peptidase CASP9 Human This gene encodes a
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID protein phosphatase, Mg2+/Mn2+ dependent, 1A PPM1A Human The protein encoded by
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Gene Information Gene Name minichromosome maintenance complex component 8 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID MCM8 Human The protein encoded by
PrimePCR Assay Validation Report
Gene Information Gene Name keratin associated protein 9-2 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID KRTAP9-2 Human This protein is a member of the keratin-associated
PrimePCR Assay Validation Report
Gene Information Gene Name SRY (sex determining region Y)-box 6 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID SOX6 Human This gene encodes a member of the
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Gene Information Gene Name keratin 3 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID KRT3 Human The protein encoded by this gene is a member of the keratin
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID protein kinase N1 PKN1 Human The protein encoded by this gene belongs to the protein
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID Peptidyl-glycine alpha-amidating monooxygenase Peptidylglycine alpha-hydroxylating
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID myeloperoxidase MPO Human Myeloperoxidase (MPO) is a heme protein synthesized during
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Gene Information Gene Name 3-hydroxybutyrate dehydrogenase, type 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID BDH1 Human This gene encodes a member of
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID peptidylglycine alpha-amidating monooxygenase PAM Human This gene encodes a multifunctional
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Gene Information Gene Name laminin, beta 3 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID LAMB3 Human The product encoded by this gene is a laminin that
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Gene Information Gene Name heat shock 10kDa protein 1 (chaperonin 10) Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID HSPE1 Human This gene encodes a major
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Gene Information Gene Name cholinergic receptor, nicotinic, alpha 9 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID CHRNA9 Human This gene is a member of
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Gene Information Gene Name transforming growth factor, beta 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID TGFB1 Human This gene encodes a member of the
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase
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Gene Information Gene Name peptidylprolyl isomerase A (cyclophilin A) Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID PPIA Human This gene encodes a member
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Gene Information Gene Name keratin 5 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID KRT5 Human The protein encoded by this gene is a member of the keratin
PrimePCR Assay Validation Report
Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID PARK2 co-regulated PACRG Human This gene encodes a protein that is conserved across
PrimePCR Assay Validation Report
Gene Information Gene Name sortilin 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID SORT1 Human This gene encodes a protein that is a multi-ligand type-1
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Gene Information Gene Name cytochrome P450, family 2, subfamily C, polypeptide 9 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID CYP2C9 Human This gene encodes
PrimePCR Assay Validation Report
Gene Information Gene Name bestrophin 3 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID BEST3 Human BEST3 belongs to the bestrophin family of anion channels
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID coagulation factor XIII, A1 polypeptide F13A1 Human This gene encodes the coagulation
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Gene Information Gene Name growth differentiation factor 6 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID GDF6 Human This gene encodes a member of the bone
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID thyroglobulin TG Human Thyroglobulin (Tg) is a glycoprotein homodimer produced
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Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID STE20-related kinase adaptor alpha STRADA Human The protein encoded by this gene
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Gene Information Gene Name ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID ATP2A2 Human This gene
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Gene Information Gene Name collagen, type II, alpha 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID COL2A1 Human This gene encodes the alpha-1 chain of
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Gene Information Gene Name glutathione S-transferase alpha 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID GSTA1 Human Cytosolic and membrane-bound forms
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Gene Information Gene Name fibroblast growth factor receptor 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID FGFR1 Human The protein encoded by this gene
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