miRNA icroRNAs – developing new tools for diagnostics · 2009. 4. 30. · miRNA icroRNAs –...

i RNA microRNAs – developing new tools for diagnostics –

Join forces with IMGM Laboratories to make your miRNA project a success

D C l WDr. Carola WagnerIMGM Laboratories GmbH

Martinsried, Germany

qPCR 2009 SymposiumMarch 10th, 2009

Agenda

• IMGM - your outsourcing partner (in brief)

• Our service portfolio (in brief)

• miRNA in diagnostics

• workflow: microarray-based miRNA profiling

• Summary

10.03.2009 © IMGM Laboratories 2009 Slide 2

IMGM Laboratories your outsourcing partnerIMGM Laboratories – your outsourcing partner


IMGM – your outsourcing partner some basic factssome basic facts

IMGM L b i f d d i 2001• IMGM Laboratories was founded in 2001

• We are located on the biotech campus pMartinsried / Munich

W t i id ff i • We operate as service provider offering advanced genomic services

• Our customers come from academia, pharma and biotech

• a flexible and reliable partnership(e.g. small and dedicated team)


IMGM – your outsourcing partner some basic factssome basic facts

• strategic partnership with the “Center for human genetics and laboratory strategic partnership with the Center for human genetics and laboratory medicine Dr. Klein & Dr. Rost”

• full accreditation according to DIN EN ISO/IEC 17025

f ll d f l b d h l • full accreditation for expression analysis based on whole genome microarrays and quantitative real-time PCR

• Agilent Certified Service Provider


IMGM Laboratories Service PortfolioIMGM Laboratories – Service Portfolio


Service Portfolio


Service PortfolioRNA servicesRNA services

RNA services:RNA services:• Microarrays (Agilent platform, multiple options)• Real-Time PCR (AB 7900HT, TaqMan arrays,

96 and 384 well blocks gene expression studies)96- and 384-well blocks, gene expression studies)

• microRNA profiling• RNA isolation

RNA l l • RNA quality control (Agilent Bioanalyzer | Nanodrop)


microRNAs and their potential in diagnostics


miRNAs in diagnostics

miRNAs / miRNA profiles:miRNAs / miRNA profiles:

• association to ~70 diseases association to 70 diseases (HMDD, human microRNA disease database, last update 2008-10-17)

• active players in human oncogenesise.g. miR-21 onco-miRNA for breast cancer (Qian et al. (2008) Breast Cancer Res Treat; c f Sassen et al (2008) Virchows Arch 452:1-10Treat; c.f. Sassen et al. (2008) Virchows Arch 452:1 10

• classify human cancerse.g. Lu et al. (2005) Nature 435:834-838Volinia et al. (2006) Proc Natl Acad Sci USA 103:2257-2261



miRNAs / miRNA profiles associated with:

• disease prognosis:− miR-221/222 cluster associated with poor prognostic in thyroid

ill i ti d i & li bl tpapillary carcinomas, pancreatic adenocarcinoma & glioblastoma(Lesage et al. (2007) Cell Cycle 6)

− Low let-7(a) expression associated with shorter survival and poor i f l ti t prognosis for lung cancer patients

(Takamizawa et al. (2004)Cancer Res 64:3753-3756; Yanaihara et al. (2006) Cancer Cell 9:189-198)

• prognosis of therapy outcome and response− high expression of miR-21 associated with poor survival and poor

h f l dtherapeutic outcome of colon adenocarcinoma(Schetter et al. (2008) JAMA 299(4):425-436)

− prediction of chemotherapeutic response in patients with breast cancer according to miRNA signature(Salter et al. 2008) PLoSOne 3(4):e1908)



Actual examples of miRNA diagnostic tests:Actual examples of miRNA diagnostic tests:

• Chronic pancreatitis vs. pancreatic cancer (Asuragen)

• Prediction of colon cancer (launch early 2009, Exiqon A/S)

• Differentiation of mesothelioma from other cancers (miRview meso, Rosetta Genomics)

• Identification of squamous-type lung cancers (miRview squam Rosetta Genomics)(miRview squam, Rosetta Genomics)

• Identification of tissue-of-origin of metastatic tumors ( )(miRview mets, Rosetta Genomics)


Possibilities for you to join

the field of miRNA research !!!the field of miRNA research !!!


miRNA profiling the general workflowthe general workflow

IMGM /Customer Agilent Bioanalyzer Catalog/Custom ArraysSample Size IMGM /Customer Agilent Bioanalyzer Catalog/Custom ArraysSample Size

Project Consulting

Sample Preparation

Quality Control

Agilent DNA Microarrays

BioinformaticData Analysis

Hit Validationwith qPCR

miRNA mimicor inhibition

Functionalanalysis

Deregulated GenesAB7900HT/TLDAsCustomere.g. mRNA microarrays


(GEx)

miRNA profilingsample preparationsample preparation

Sample types:• Cells

Ti• Tissues• Blood and PBMCs• FFPE

e.g. Li et al. (2007) BMC Biotechnology 7:36Xi et al. (2007) RNA 13:1668-1674Siebolts et al. (2009) J Clinc Path 62(1):84-88

• Body fluids e g serum urine salvia amniotic fluid pleural fluide.g. serum, urine, salvia, amniotic fluid, pleural fluide.g. Chen et al. (2008) Cell Research 18:997-1006Gilad et al. (2008) PLoS One 3 (9):3148Hunter et al (2008) PLoS One 3 (11):e3694


Hunter et al. (2008) PLoS One 3 (11):e3694

miRNA profiling(mi)RNA quality control & quantification(mi)RNA quality control & quantification


www.agilent.com/chem/labonchip


Tissot (2008) Agilent Application Note, Publication Number 5989-7878EN; www.agilent.com/chem/laponchip

miRNA profilinge g Agilent Microarray Workflowe.g. Agilent Microarray Workflow

Total RNA incl. small RNAs(100 )(100ng)

direct RNA 3’end Labelingwith Cy3-pCp

Labeled RNAs

hybridization on microarrays

miRNA profile


miRNA profiling Data analysisData analysis


Technical Note 15 (3), www.ambion.com Yanaihara et al. (2006) Cancer Cell 9(3):189-198

miRNA profiling Data analysisData analysis

Sample treated log2 data

Collaborative work with SIRION BIOTECH:Thirion et al. (2009) qPCR 2009 – RNAi:

microRNA – siRNA applications

S l d


Sample_untreated log2 data

miRNA profiling Hit validation by qPCRHit validation by qPCR

6

* ** * **linear Fold

2

4

Fold Change

2

‐2

0

calibrator miR‐103

calibrator miR‐27a

calibrator miR‐23b + miR_103

-2

‐6

‐4 Collaborative work with SIRION BIOTECH:Thirion et al. (2009) qPCR 2009 – RNAi:

microRNA siRNA applications

‐8

6

* p<0.05

microRNA – siRNA applications


‐10p

** p<0.01

miRNA profiling e g functional analysis by mRNA expression profilinge.g. functional analysis by mRNA expression profiling

miRNA overexpression miRNA

inhibition inhibition mRNA profiling

= gene expression

analysis


www.thco.com.tw

Summary

Single miRNAs / miRNA profiles in relation to diagnostics:• Well established miRNA technologies• Increased number of publications with promising results

Fi t di ti t t l h d • First diagnostic tests launched

miRNA research for you powered by IMGM laboratories:miRNA research for you powered by IMGM laboratories:• Advanced genomic services with one focus on RNA services and miRNA

services• State-of-the-art technology to provide top-quality data• Full accreditation – especially for microarray- and qPCR-based expression

analysisanalysis• Sound bioinformatics to extract meaningful results• Expert knowledge in molecular genetics and medical diagnostics


Thank you very much for your attention!

We are open for business…

The IMGM Laboratories TeamD. Wiegand, M. Hirt, S. Brauer, S. Kotschote, H-G. Klein, R. Oehlmann, C. Wagner, M. Sohns

www.imgm.com


miRNA icroRNAs – developing new tools for diagnostics · 2009. 4. 30. · miRNA icroRNAs –...

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