somatic mutation profiling and mirna discovery in cancer...

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


• 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

Title, Location, Date 4

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


Advances in sequencing technology leading to a significant increase in the number of known somatic mutations involved in cancer:



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


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


QIAamp DNA Mini KitQIAamp DNA FFPE Tissue

Kit


*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


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


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%


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


NOTE: Corresponding qBiomarker Probe Master Mixes are included with ALL ORDERS.


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


microRNA - Discovery tool, potential therapeutic

Sample & Assay Technologies What is microRNA?


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


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


Title, Location, Date

miScript miRNA PCR Workflow

33

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


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


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


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


www.qiagen.com/geneglobe


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


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



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



Sample & Assay Technologies PCR Arrays for Disease Research


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


Many Scientists have discovered the power of PCR Arrays.Join them on the road of success!


• 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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somatic mutation profiling and mirna discovery in cancer...

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