Sample & Assay Technologies
Somatic Mutation Profiling and miRNA discovery in Cancer Research
Annie Chan D.Phil. (Oxon)Assoc. Regional Marketing Manager, Asia Pacific
Sample & Assay Technologies QIAGEN - Complete Solution For Your Application
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Sample preparation(stabilization, disruption,...)
RNAlaterQIAzol
QIAmpRNeasyDNeasymiRNeasyAllprotect
TissueLyser II TissueLyser LT QIAgility Rotor-Gene QQIAcube
RNA Purification & cDNA Synthesis
Assay SetupReal-time PCR analysis
qBiomarker Somatic MutationPCR Array & Assays
miScript miRNA PCR Array & Assays
+GeneGlobe Web Portal
For Internal Use Only
Data Analysis Tools
Sample & Assay Technologies
• SABiosciences is now a Company
Research Tools for disease and pathway profiling
qBiomarker Somatic Mutation PCR ArraysReal-time PCR based methods
miScript miRNA PCR ArraysReal-time PCR based methods
Sample & Assay Technologies Methods for Genetic Analysis
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Instrumentation & reagent cost
Number of biological sample to be processed in one run
Microarray• Profile More Sequences• Poor Sensitivity & Dynamic Range and
complicated Protocol
Sequencing• High capital cost• Required third party software
/sophisticated bioinformatics analysis
Real-time PCR (qPCR)• Fast and Simple Protocol • Better Sensitivity, Specificity & Reproducibility• Ease of use and maintenance• Well established technology
Sample & Assay Technologies Agenda
miScript PCR System
Technological Principles
miRNA – Discovery Tool to Therapeutic
What is a qBiomarker Somatic Mutation PCR Array
Workflow and Analysis
Background
Array Performance and Data
Summary
qBiomarker Somatic Mutation PCR Array
GeneGlobe Web Portal
Sample & Assay Technologies What is a Somatic DNA Mutation?
� Definition: � Somatic Mutation vs Germ Line Mutation, NOT passed on to
children� Single Point Mutation or Multiple Mutations Possible within a
Gene
� Causes of Somatic Mutation:� Copying Error as part of DNA Replication
– Estimated error rate of DNA polymerase: 1 in108 bases� DNA damage due to external factors
– e.g. UV Exposure, Radiation, Cigarette Smoke
Sample & Assay Technologies Translating Somatic Mutations into Therapeutic Benefits
� ~ 140,000 DNA Mutations Identified
� Multiple Consortiums
� COSMIC: Catalog of Somatic Mutations in Cancer: Sanger Institute
� HGMD: The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff
� dbRBC: Blood Group Antigen Gene Mutation Database: NCBI
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Advances in sequencing technology leading to a significant increase in the number of known somatic mutations involved in cancer:
Sample & Assay Technologies
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Therapeutic Benefits of Somatic Mutations
Personalized Medicine• Where clinicians treat patients based on their genotype
or somatic mutations• For example, drug development and toxicology studies
Prognosis • Identification of genotypes that correlated with clinical
outcomes
Molecular mechanisms study• Basic research to link mutations with various
disease/pathway
Translating Somatic Mutations into Therapeutic Benefits
Sample & Assay Technologies
Method Benefits Drawbacks
Point Mutation Analysis Exact Time Consuming
Quantify DNA Targets� Real-time PCR
FastReliableHigh-throughput
Mass Spectrometry Exact Time ConsumingNeed Instrument/Training
High Resolution Melt InexpensiveFastSimple
Base change identity not obvious
Microarray Large Coverage SensitivityProtocol complexNeed Instrument/Training
Next-Generation Sequencing
Exact Time ConsumingNeed Instrument/Training
Analysis Methods for DNA Mutations
Sample & Assay Technologies What is the Somatic Mutation PCR Array?
EGFR Pathway
• EGFR – 29 Mutations
• AKT1 – 1 Mutation
• BRAF – 12 Mutations
• KRAS – 18 Mutations
• HRAS – 11 Mutations
• NRAS – 12 Mutations
• MEK1 – 4 Mutations
• PI3K – 7 Mutations
• PTEN – 6 Mutations
Collection of mutation specific assays for multiple mutations in a single gene, as well as mutations present in genes in downstream signaling activities
Sample & Assay Technologies Technological Principles
• Relies on the 5’-3’ exonuclease activity of the Taq DNA Polymerase
• Reporter dye signal is quenched by the proximity of a Quencher dye
• Exonuclease activity hydrolyses the probe to liberate the Reporter from Quencher
• End result is the detection of signal from the Reporter dye
The qBiomarker Somatic Mutation PCR Assays contain panels of bench-verified hydrolysis probe-based real-time PCR assays
F Q
Common Region
Sample & Assay Technologies Key to qBiomarker Somatic Mutation Assay Design
Amplification Refractory Mutation (ARMS) System technology
Strategy in which Primers discriminates amongst DNA Templates that differs by a Single Nucleotide
• ARMS Primers are designed to match mutation sequence
• Taq DNA Polymerase will bind and initiate the PCR reaction
• ARMS primers do not match wild-type DNA sequence
• Taq DNA Polymerase will not bind and initiate PCR reaction
Sample & Assay Technologies Fast and Simple Workflow
Purification30 mins
Detection2 hrs
Analysis10-20 mins
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QIAamp DNA Mini KitQIAamp DNA FFPE Tissue
Kit
qBiomarker Somatic Mutation PCR Array
*Corresponding qBiomarker Probe Master Mixes are included with the array
Analysis Template available online
The status of up to 85 mutations can be screened in less than 3 hours
Sample & Assay Technologies
Fresh/frozen samples: 200 – 500 ng gDNA
FFPE samples: 200 – 500 ng gDNA
Fresh/frozen samples: 5 – 10 ng gDNA
How does the Somatic Mutation PCR Array Work?
Sample & Assay Technologies Anatomy of a Somatic Mutation PCR Array
Greek Symbol = Gene
Number = Mutation
• For normalization
• Assays detect non-variable region (not ARMS-based design)
• Designed to control differences in sample input and quality
Sample & Assay Technologies Principles of Data Analysis
Note: The qBiomarker Somatic Mutation PCR Arrays shows any presence of a mutation. It only presents a qualitative analysis and not quantitative information.
•Purpose: Qualitative assessment of samples
• Is the DNA Mutation Present and if so, which one?
• Data Analysis Methods depends on:
• 1. Availability of wild-type sample
• 2. Sample type/quality
• Cell lines
• Fresh or frozen samples
• FFPE samples
• 3. How many samples?
Two methods (published in white paper)
•1. ∆∆CT method
•2. Average CT method
Sample & Assay Technologies Principles of Data Analysis - ∆∆ Ct
∆ Ct Control Sample
∆ Ct Test Sample
Average ∆ Ct Control Sample
Step 1
Step 2 Step 3
Step 4
∆∆ Ct
Sample & Assay Technologies Principles of Data Analysis - Average Ct
Sample & Assay Technologies
Data Analysis Method Selection Summary• ∆∆Ct method
• Must have wild-type control• Works well for projects using:
• Fresh/frozen samples• Smaller (≤4) number of samples
• i.e. sample quality similar
• Average Ct method• Works well for projects using:
• FFPE samples• Large number of samples
• i.e. sample quality can be in a wide range
• Without Wild-type controls
qBiomarker Somatic Mutation PCR Array Data Analysis
Two Methods: ∆∆Ct vs Average Ct: How to Choose
Sample & Assay TechnologiesqBiomarker Somatic Mutation PCR Array Data Analysis∆∆Ct Method
Recommended for experiments using:
• Small #s of samples (< 4)
• Basic Cell Line Experiments….Looking for Mutation in just a couple of cell line samples
• Results affected by sample quality (Wild-type and Test Samples)
• Wild-type controls NECESSARY
• Wild-type controls MUST be of high-quality
• Considers Copy # Changes
• NORMALIZES (∆Ct)
• Caveats
• Penalizes good quality samples (False Negatives)
• Rewards bad quality samples (False Positives)
Sample & Assay TechnologiesqBiomarker Somatic Mutation PCR Array Data AnalysisAverage Ct Method
Recommended for experiments using:
• FFPE samples
• Large #s of samples (> 4)
• Wide range of sample quality
• Wild-type controls not necessary
• Assumes that for a given locus, mutation occurs in small % of samples.
∴Ctaverage
= Mutation Assay Background in Wild-Type Sample
�Compare Ctsample
to Ctaverage
= ∆Ct
� If Ctsample
<< Ctaverage
� Sample has Mutation
Sample & Assay Technologies Assay Performance – Differentiation between Mutant and Wild-Type
Assay differentiation window (ADW). The ADW was defined as the CT difference between the signals generated on a wild-type genomic DNA background and on 100% mutant template by a mutation assay. Assays were verified to have a minimal ADW of 8 but are usually higher.
Sample & Assay Technologies Assay Performance – Sensitive Detection of low % mutations
Assay sensitivity test for p53 R280K qBiomarker Som atic Mutation Assay.
Sample: 10 ng genomic DNA samples with 4, 21, 106, 528, 2640 and 13200 copies of mutant DNA template respectively, from MDA-MB-231 cell line (mutant harboring the p53 R280K mutation) mixed with genomic DNA from the Coriell GM00131 cell line (wild-type).
Method: qBiomarker Somatic Mutation Assay for p53 R280K Assays were performed in duplicate.
Results:Mutation detection limit for this assay is determined to be 0.03%. Amplification efficiency is close to 100%
Sample & Assay Technologies
Samples: 10 Asian, Female, non-smoker origin; FFPE Lung Adenocarcinoma
Method: Human EGFR Pathway qBiomarker Somatic Mutation PCR Array
Results: 9 mutations, including 1 BRAF mutation in 1 sample, 4 EGFR mutations in 3 samples and 4 KRAS mutations in 4 samples
Mutations
FFPE Samples
KRAS∆Ct
BRAF
EGFR
Detection of BRAF, EGFR, and KRAS Mutations in FFPE samples
Sample & Assay Technologies qBiomarker Somatic Mutation Assay Validation
Pyrosequencing Confirmation of KRAS & EGFR Mutations
Sample & Assay Technologies
NOTE: Corresponding qBiomarker Probe Master Mixes are included with ALL ORDERS.
qBiomarker Somatic Mutation PCR Array
Simultaneous Profiling of Gene Mutations within a Disease or Pathway
Cancers Pathway
Brain Cancer APC/CTNNB1
Breast Cancer c-MET
Colon Cancer ErbB2
Hematopoietic Neoplasms EGFR
Lung Cancer FGFR
Lymphoid Neoplasms FLT3
Melanoma KIT
Skin Cancer p53/Rb
Soft Tissue Tumors PDGFR
Thyroid Cancer PI3K-PTEN
Bladder Cancer Ras-Raf
Endometrial Cancer RTK Panel I
Liver Cancer RTK Panel II
NEW! Ovarian Cancer
NEW! Gastric Cancer
Pancreatic Cancer
“… …the qBiomarker Somatic Mutation PCR Array System is designed for rapid and accurate mutation profiling in basic research a nd drug discovery… …”
Science, 7 October 2011: Vol.334 No.6052 Pg.113 DOI:10.1126/science.334.6052.113-a
Sample & Assay Technologies
microRNA - Discovery tool, potential therapeutic
Sample & Assay Technologies What is microRNA?
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Endogenously expressed small functional RNAs (19-25 nt)
Regulate mRNA expression post-transcriptionally
Leading to translational inhibition
Only partial sequence complementarity required
One miRNA can potentially regulate hundreds of mRNAsOne mRNA can be regulated by multiple miRNAs
Another layer of complexity for regulating gene expression
Sample & Assay Technologies Role of miRNA in Human Cancer
� In 2005: Unique miRNA signatures are found in human cancer� miRNAs located in genomic regions amplified in cancers (e.g.
miR-12-92 cluster) can function as oncogenes, � whereas miRNAs located in portions of chromosomes deleted in
cancers (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.
� In 2012: A growing picture of miRNA dysregulation in cancer� a substantial number of studies and reviews have associated the
presence of various miRNAs with cell proliferation, resistance to apoptosis, and differentiation in cancer cells..
e.g. non coding RNA DLEU2 is often deleted in lymphocytic leukemia. Extensive effort at figuring out how this ncRNA was involved in this disease was unsuccessful. Recently it was discovered that there are two miRNAs encoded in the intron of this gene and it’s the deletion of these miRNAs that contributes to cancer.
The race was on to develop miRNA biomarkers and therapeutics!
Sample & Assay Technologies miRNA as ‘drug’, miRNA as ‘target of drug’
Google “miRNA drug” >50,000 hits
Sample & Assay Technologies Methods for miRNA Detection Analysis
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Instrumentation & reagent cost
Number of biological sample to be processed in one run
Microarray• Profile More Sequences• Poor Sensitivity & Dynamic Range and
complicated Protocol
Sequencing• High capital cost• Required third party software
/sophisticated bioinformatics analysis
Real-time PCR (qPCR)• Fast and Simple Protocol • Better Sensitivity, Specificity & Reproducibility• Ease of use and maintenance• Well established technology
Sample & Assay Technologies New miRNA detection portfolio: The microRNA workflow
Rotor-Gene QQIAgility
QIAcube
NEW! miRNeasy Micro Kit miScript II RT Kit HiPerFect Transfection Reagent
NEW! miRNeasy Serum/Plasma Kit miScript SYBR Green PCR Kit miScript miRNA Mimics
miRNeasy Mini Kit miScript miRNA PCR Arrays* miScript miRNA Inhibitors
miRNeasy 96 Kit miScript miRNA Data Analysis Tool miScript Target Protector
miRNeasy FFPE Kit miScript Primer AssaymiScript miRNA Inhibitor 96 and 384 Plates and Sets
miRNeasy Protect Animal Blood Kit miScript Precursor AssayCustom miScript miRNA Mimics and Inhibitors
PAXgene Tissue miRNA Kit miScript PCR Starter Kit Cignal Report Assays
PAXgene Blood miRNA Kit * System is compatible with most cycler
Purification Analysis Function
Pro
filin
g
Sample & Assay Technologies
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miScript miRNA PCR Workflow
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miRNA isolation with RNeasy kits
cDNA Synthesis from miRNA1 hours
Load Plates (Preferably with 8-Channel Pipettors)
2 minutes
Run 40 cycle qPCR Program 2 hours
Upload and analyze Data15 minutes
Sample & Assay Technologies Universal miRNA Reverse Transcription
TTTTTTTT
miRNA
AAAAAAAA
NNTTTTTTTTmiRNA RT Primer
Reverse Transcriptase
Poly(A) Polymerase
Real-Time PCR
TTTTTTTT
miRNA-Specific Primer
Universal Primer
Same cDNA preparation can assay ANY miRNA
Ease of Universal Reverse Transcription Reaction� Simpler setup, without
primer interaction issues� Less sample necessary for
analyses� Equal RT reaction for each
miRNA, to ensure reproducible expression analysis
Comprehensive miRNA coverage� cDNA can be saved for new
analyses when additional miRNA sequences are discovered
Similar potential specificity, & additional flexibility!
Sample & Assay Technologies miScript miRNA Array format
e.g. Standard 96 well plate
Controls
Cel-miR-39
miScript PCR Controls for Normalization
miRTC PPCSNORD61; SNORD68; SNORD72 SNORD95; SNORD96A; RNU6B
RTControl
PCRControl
Spike in Control
H1 2 3 4 5 6 7 8 9 10 11 12
miScript PCR Controls for normalization: Common for Human, Mouse, Rat and Dog Arrays
Biologically R
elevantm
iRN
A prim
er assays
Sample & Assay Technologies miScript miRNA PCR Arrays
� Human miScript miRNome miRNA PCR Array� Mouse miScript miRNome miRNA PCR Array� Rat miScript miRNome miRNA PCR Array� Dog miScript miRNome miRNA PCR Array� New! Rhesus Macaque
� Human, Mouse, Rat:� Brain Cancer � Breast Cancer� Cancer Pathway Finder� Cell Differentiation & Development� Immunopathology � Inflammatory Response and
Autoimmunity � miFinder� Neurological Development & Disease � Ovarian Cancer� Serum & Plasma� miRNA QC
Complete miRNA genome (miRNome) miRNA Pathway Arrays
SpeciesNumber of
assays(miRBase V16)
Total number of plates
96-well format
384-well format
Human 1068 12 3
Mouse 947 12 3
Rat 653 8 2
Dog 277 4 1
Upcoming! miRBase v18
Custom miRNA ArraysHuman, mouse, rat, dog, rhesus macaque,
and other species
Sample & Assay Technologies
You don’t own any real-time PCR Cycler
miRNA PCR Array Service Core
.96-Well Blocks: 7000, 7300, 7500, 7700, 7900HT, Vii A 7
.FAST 96-Well Blocks: 7500, 7900HT, Step One Plus, V iiA 7
.FAST 384-Well Block: 7900HT, ViiA 7
.iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384
.Opticon, Opticon 2, Chromo 4
.Mastercycler ep realplex 2/2S/4/4S
.LightCycler 480
.Mx3000p, Mx3005p, Mx4000p
.Rotor-Gene Q, Rotor-Gene 6000
Open Platform: Compatible with major real-time PCR cyclers
.TP-800
Sample & Assay Technologies User Friendly Web-based Data Analysis Tool
Free Web-Based Data Analysis Software� No installation or licensing needed
From Raw Ct Values to Fold Change Results� Using ∆∆C(t) Method
Multiple Analysis Formats� Scatter Plot� Volcano Plot� Multi-Group Plot� Clustergram
Also available as downloadable Excel templates
For Internal Use Only
FGFR4
MMP9
ETV4MMP10
MMP2
MMP3
MMP7
UP REGULATEDDOWN REGULATEDVolcano PlotLog 2 Fold Change
Log
10(P
-val
ue)
Scatter Plot
Sample & Assay Technologies Application Data: Cancer Biomarkers
miScript Human Cancer Pathway Finder PCR Array :Identification of Cancer-Specific miRNA Upregulation in Colon Tumor
Sample: small RNA isolated from human colon tumor & matched adjacent normal tissueMethod : miScript Human Cancer Pathway Finder PCR ArrayResults : More than 22 cancer-related miRNAs involved are upregulated in colon tumor sample
Sample & Assay Technologies Application Data: Genome-Wide Screening
miScript Human Genome miRNA PCR Array: Identifies Known and Novel miRNA Targets of the p53 Signaling Pathway
15
20
25
30
35
40
15 20 25 30 35 40Ct Control
Ct A
den
o-p
53
34a 940
203 551a
Cell Line : PC3Procedure: Overexpression of adenovirus-p53 vector or empty vector. Small RNA was collected 48h later.Method : Human miRNome miRNA PCR ArrayResults : (left) 4 novel miRNA targets of p53 pathway identified. (right) red, up-regulated miRNA; green, down-regulated miRNA; black, no difference
Sample & Assay Technologies
miRNA � miRNA Detection Assays & Arrays� miRNA mimics & inhibitors� miScript Target protectors
Next Generation Sequencing� SeqTarget Primer Select
siRNA� FlexiTube siRNAs & Premix� FlexiPlate � shRNA plasmids
DNA methylation� PyroMark CpG Assays� Epitect Methyl Profiler Arrays & Assay
Chromatin IP� Epitect Chip Assays & Arrays
GeneGlobe: combination of gene and pathway selection tool
qPCR/Gene expression� QuantiTect Primer Assays� QuantiFast Probe Assays � RT2 PCR Arrays & Assays
Gene expression
siRNAmiRNA
Epigenetics
NGS
Cell signaling
Somatic Mutation � qBiomarker Somatic Mutation Array & Assay
CIGNAL Reporter � CIGNAL Reporter Arrays & AssayGenotyping
Sample & Assay Technologies GeneGlobe Web Portal
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www.qiagen.com/geneglobe
Sample & Assay Technologies
Choose “Tube” or “Plate”
Select your products(s)
1 Go to QIAGEN GeneGlobe on www.qiagen.com/geneglobeEnter GeneID, Ref Seq ID or Cat.No. When using gene symbols or names please select species. Press “Search“ button.
2
3
B
A
Get a first search result, including an overview which products are available for your gene(s) of interest, and which pathways are associated to the genes. Click on "View products“ or on the respective tick to see the products.
QIAGEN GeneGlobeThree steps to select your QuantiTect Primer Assay
Sample & Assay Technologies Gene Details
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Sample & Assay Technologies Product Details
Sample & Assay Technologies QIAGEN GeneGlobe Web Portal
46
QT Primer Assay detail page – upper part
� Amplicon location� Coding Sequence� SNP
Sample & Assay Technologies QIAGEN GeneGlobe Web Portal
47
QT Primer Assay detail page – lower part
Links to external databasee.g. Entrez Gene, ENSEMBL
Amplicon details
Sample & Assay Technologies Pathway Information
Sample & Assay Technologies Example human TP53 – Entrez ID 7157
Pathway imageAll genes of the respective pathway
Sample & Assay Technologies Interactive Pathway Maps for Ordering
Interactive pathway based information and ordering system
Sample & Assay Technologies GeneGlobe Web Portal
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www.qiagen.com/geneglobe
Sample & Assay Technologies Pathway Central: Example Akt
Download image or Powerpoint slide
Interactive image – to select genes one by one
Sample & Assay Technologies Products via Pathways
Unique ability to select more than one gene
Gene details like Acc.No etc.
Select here the genes and add them tothe „Your gene selection list“ Click here to find the
products for selected gene
Sample & Assay Technologies Pathways details
Interactive linksWill add genes to „Your gene selection“
Interactive reference listlinks to PubMed
Sample & Assay Technologies Need more information?
GeneGlobe Web PortalExplore the gene- and pathway-specific products
www.qiagen.com/geneglobe
For Internal Use Only
Sample & Assay Technologies PCR Arrays for Disease Research
For Internal Use Only
PCR Arrays for disease research is now added in GeneGlobe
Sample & Assay Technologies Need more information?
Webinar (Live and Recorded)http://www.qiagen.com/events/webseminarshttp://www.sabiosciences.com/seminarlist.php
Video: How a miScript miRNA PCR Array works– http://www.sabiosciences.com/mirna
Contact:[email protected] 930 425Contact Local Sales Representative
Sample & Assay Technologies Upcoming webinar
Registration Link https://www2.gotomeeting.com/register/836153578
Sample & Assay Technologies SUMMARY
Both Somatic Mutation and miRNA are important factors in Cancer Research while QIAGEN provides the research tools to facilitate your study
Fast and Simple Workflow � 80-350 cancer mutations per sample in a single PCR run (qBiomarker Somatic Mutation PCR
Array)
� 376 miRNA in a single PCR run for human miRNA profiling, whole human miRNAgenome in 3x 384 plates or 12x96 plates (miScript miRNome PCR array)
Superior Detection Sensitivity� As low as 0.01% mutant DNA in a wild-type background (for qBiomarker Somatic
Mutation PCR Array)� As low as 10 copies of miRNA per reaction (for miScript PCR System)
Reliability� Assays are web-bench validated and biological relevant in a pathway/disease
Flexibility to use� Applicable to almost all sample type, e.g. fresh/frozen cells and tissue, FFPE,
serum/plasma samples� Customizable and compatible with all major qPCR instrument
Sample & Assay Technologies
Many Scientists have discovered the power of PCR Arrays.Join them on the road of success!
Sample & Assay Technologies
• qBiomarker Somatic Mutation PCR Array and miScript miRNA PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
•For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
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