Mi rna part i_2013

Sample & Assay Technologies

Meeting the challenges of miRNA research :

microRNA Biogenesis, Function, and Analysis

Jonathan Shaffer, Ph.D.

[email protected] Technologies, R&D Americas

The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of disease.

Sample & Assay Technologies Three part webinar series

Webinar 1: Meeting the Challenges of miRNA Research 2

miRNA and its role in human disease

Webinar 2 : Advanced microRNA expression analysis: From experimental design through data analysis

Speaker: Jonathan Shaffer, Ph.D.

Webinar 3 : Profiling miRNA expression in Cells, FFP E, and serum:On the road to biomarker development


Webinar 1 : Meeting the challenges of miRNA research :An introduction to microRNA biogenesis, function, a nd analysis


Sample & Assay Technologies Upcoming webinars


April 2013

Sign up at http://www.SABiosciences.com/seminarlist .php

Sample & Assay Technologies Meeting the Challenges of miRNA Research


Agenda

� miRNA Background

� miRNA Genomics

� miRNA in Disease

� miRNA Isolation Technologies

� miRNA Quantification Technologies

� miRNA Profiling Technologies

� miRNA Functionalization Technologies

Sample & Assay Technologies RNA interference: A natural phenomenon


Discovery tool, potential diagnostic, potential the rapeutic

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� Changes in miRNA can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging, and disease.

Sample & Assay Technologies Canonical pathway of miRNA biogenesis


� Transcribed by RNA Polymerase II as a long primary transcript (pri-miRNAs), which may contain more than one miRNA.

� In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha.

� Pre-miRNAs are then exported to the cytosol by Exportin 5.

� In the cytosol RNAse III-like Dicer processes these precursors to mature miRNAs.

� These miRNAs are incorporated in RISC.

� miRNAs with high homology to the target mRNA lead to mRNA cleavage.

� miRNAs with imperfect base pairing to the target mRNA lead to translational repression and/or mRNA degradation.

Sample & Assay Technologies How do miRNAs interact with mRNAs?


Basis of miRNA-mRNA interaction

Seed region: nucleotides 2-8 in 5’ region of miRNA� Most evolutionary conserved miRNA region� Most frequently complementary to target 3’-UTRs� Often sufficient to confer mRNA recognition

Beyond the seed region� 3’ end also contributes (extensive pairing is rare)� Some cases: central 11-12 continuous base pairs

Result of interaction� Suppression of gene expression� Rare cases: increase gene expression

References� Grimson, A., et al, Mol. Cell 2007, 27, 91-105 � Image From Bartel, D.P., Cell 2009, 136, 215-233� Guo, H., et al, Nature 2010, 466, 835-840� Thomson, D.W., et al, Nucleic Acids Res 2011, 1-9

Sample & Assay Technologies How to determine miRNA-mRNA interactions


Algorithms for predicting miRNA-mRNA interaction

Prediction Algorithm Website

TargetScan http://www.targetscan.org/

Pictar http://pictar.mdc-berlin.de/

MicroCosm Targets http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/

DIANA http://diana.cslab.ece.ntua.gr/microT/

miRANDA http://www.microrna.org/microrna/home.do

TarBase (experimentally supported)

http://diana.cslab.ece.ntua.gr/tarbase/

Target Prediction is based on:� Bioinformatics

� Seed region match� Position in 3’ UTR� Cross species conservation� Central sequence homology

� Wet lab research� Empirical evidence from microarrays� Reporter systems

Pitfalls of using prediction algorithms:� Large number of candidate mRNAs for a given

miRNA� May not incorporate all miRNA targeting

possibilities� Different algorithms produce different target

lists� Potential for false positive rate of prediction

Sample & Assay Technologies How to determine miRNA-mRNA interactions


Experimental techniques

miRNA Target Screening� Gene expression analysis (inferred targets)

� RNA-seq� Microarrays� qPCR

� Immunoprecipitation (direct targets)� HITS-CLIP� PAR-CLIP� Biotin tagged miRNA

Gene-Specific Validation� qPCR� Luciferase reporter assays� Western blot� 5’ rapid identification of cDNA ends (5’ RLM-RACE)

Other techniques� Parallel analysis of RNA ends (modified 5’ RLM-RACE)� Reverse transcription of targets

Image From Chi, S.W., et al, Nature 2009, 13, 479-486



miRNA Genomics: Generating miRNome Diversity

Functionalization

Isolation

Quantification and Profiling

Sample & Assay Technologies miRNA genomic structure


� Intergenic miRNA genes : either monocistronic or polycistronic with a common promoter

� Intronic miRNA genes : present in the introns of protein coding or noncoding genes, can also be in clusters, transcribed by the host gene promoter

� Exonic miRNAs genes : rare and often overlap an exon and an intron of a noncoding gene

� miRNAs can be transcribed from the negative strand within or near a protein coding gene

Sample & Assay Technologies Multiple loci can generate the same mature miRNA


Biogenesis creates incredible diversity

www.mirbase.org

Stem Loop CHR Overlapping transcripts CHR: Coordinat es (GRCh37)

1302-1 12 intergenic 12: 113132839-113132981 [-]

1302-3 2 intergenic 2: 114340536-114340673 [-]

1302-7 8 intergenic 8: 142867603-142867674 [-]

1302-10 15 intergenic 15: 102500662-102500799 [-]

1302-11 19 intergenic 19: 71973-72110 [+]

1302-2 1 intronic Non protein coding 1: 30366-30503 [+] sense

1302-4 2 intronic Non protein coding 2: 208133999-208134148 [-]

1302-9 9 Non protein coding 9: 30144-30281 [+]; Sense

1302-5 20 intronic Protein coding/FAM65C; intron 4 20: 49231173-49231322 [-]; Sense

1302-6 7 intronic Protein coding/HDAC9; intron 1 7: 18166843-18166932 [-] ; Antisense

1302-8 9 intronic Protein coding/ch9orf174 9: 100125836-100125963 [-]; Antisense

Mature-miR-1302: UUGGGACAUACUUAUGCUAAA

Sample & Assay Technologies Post-transcriptional modification of miRNA


Processing and ADAR editingD

eep

sequ

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f hsa

-miR

-21-

5p

Pre-miRNA

Exportin

Pri-miRNA

ADAR

A�I

A��I DROSHA Processing Block

A��IDICER

Processing Block

Change in miRNA seedA��I

ADAR

ADAR

ADAR: adenosine deaminase, RNA specificA-> I destabilizes dsRNA



miRNA in Disease

Functionalization


Isolation

Sample & Assay Technologies Potential events that disrupt normal miRNA activity

15

Disruption of miRNA-mRNA interaction

Webinar 1: Meeting the Challenges of miRNA Research

Altered TranscriptionMethylation

Histone ModificationTranscription Factor

Drosha Processing

Genomic InstabilityAmplification/DeletionTranslocationInsertional Mutagenesis

Loss of miRNA Binding Site in targetSNP or MutationAlternative SplicingLoss/Change of 3’-UTR

Dicer Processing

Sample & Assay Technologies Unique signatures in human cancer


� miRNAs located in genomic regions amplified in cancers (e.g. miR-17-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cance rs (e.g. miR-15a-miR-16-1 cluster) can function as tumor suppressors.

� Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors.

� miRNA expression fingerprints correlate with clinical and biological characteristics of tumors , including tissue type, differentiation, aggression and response to therapy.

In the last 10 years, a substantial number of studi es and reviews have associated the presence of various miRNAs with cell proliferation,

resistance to apoptosis, invasiveness, and differen tiation in cancer cells.

Sample & Assay Technologies miRNA in cancer diagnosis


Paranjape et al. (2009)

Gut. 58: 1546-1554.

Utilizing a signature of altered miRNA expression to

differentiate cancer tissue from normal tissue

Profiling circulating blood or tumor derived exosomalmiRNAs, surpassing the

invasive procedures to aid in early detection of cancers

Study SNPs in the miRNA genes, miRNA binding sites in the target

mRNA genes or in the miRNA processing/machinery pathway genes

to predict cancer predisposition

Use of miRNA based classifier to identify tissue

of origin for cancers of unknown primaries

Distinguishing tumor subtypes using a panel of

miRNAs that show differential expression within one cancer type

miRNAs in Cancer Diagnosis



miRNA Isolation Technologies

Functionalization

Isolation


Sample & Assay Technologies miRNeasy Kits: Mini, Micro


Flexible protocols: total RNA or enrichment of miRN A

.High quality, pure RNA � Suitable for sensitive downstream

applications

.Rapid procedure � Streamlined protocol for low-

throughput or 96-well formats� Automated on QIAcube

Effective purification of total RNA� From a broad range of cells & tissues

.Efficient enrichment of microRNAs� miRNA enriched fraction & total RNA

separately� Co-purification of miRNA & total RNA

Sample & Assay Technologies miRNeasy FFPE Kit


High quality total RNA from archival samples

500 million FFPE tissues are archived!� Tissue banks, pathology labs, biomedical research labs

Current Isolation Methods: Compromised quality and yield� Heavily fragmented� Cross-linking by formaldehyde interferes with RT� Current procedures may not purify all usable RNA, may increase

fragmentation, are often ineffective in breaking up cross-links

.miRNeasy FFPE Kit: High quality, pure total RNA� Novel method prevents cross-linked RNA from blocking

downstream applications� Optimized lysis buffer with proteinase K� RNeasy MinElute for small elution volume

Sample & Assay Technologies miRNeasy Serum/Plasma Kit


Purification from serum, plasma, and other fluids

� Includes synthetic RNA control assay for normalization

� Minimal elution volume (14 µl)

� High-purity RNA suitable for all downstream applications� miScript RT: up to 10 µl eluate for cDNA synthesis� One RT enough for 6, 384-well plates

� Easy, robust procedures

� Automatable protocol

QIAzol Bind Wash Elute

Manual or Automated on QIACube

Plasma Serum

Clarified plasmaor serum

For purification of circulating RNA from animal and human plasma and serum



miRNA Quantification Technologies

Functionalization

Isolation


Sample & Assay Technologies miScript PCR System


Fully integrated, complete miRNA quantification sys tem

1. miScript II RT Kit� HiFlex Buffer: Unparalleled flexibility for miRNA and

mRNA quantification from a single cDNA preparation� HiSpec Buffer: Unmatched specificity for mature

miRNA profiling

2. miScript miRNA PCR Arrays� miRNome� Pathway-focused

3. miScript PreAMP Kit� Optional step for small or precious samples� Full miRNome profiling from as little as 1 ng RNA

4. Assays� miScript Primer Assays� miScript Precursor Assays� QuantiTect Primer Assays

5. miScript SYBR Green PCR Kit� QuantiTect SYBR Green PCR Master Mix� Universal Primer

6. miScript miRNA PCR Array data analysis software � Straightforward, free data analysis

Sample & Assay Technologies miScript II RT Kit


A complete miRNA quantification solution

miScript II RT Kit

Biogenesis studies?Mature miRNA

quantification and profiling?

HiFlex Buffer HiSpec Buffer

Flexible detection of all RNA molecules

Patent-Pending technology for the specific detection of mature miRNAs

Note: Only HiSpec Buffer is recommended for use with miScript miRNA PCR Arrays

Sample & Assay Technologies HiFlex Buffer or HiSpec Buffer:


Which buffer should you choose?


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Experiments that demand quantification of both mature miRNAs & mRNAs.

Experiments designed exclusively for mature miRNA quantification. Long RNAs, such as mRNAs, are not converted into cDNA. HiSpec is the optimized buffer for mature miRNA profiling with miScript miRNA PCR Arrays.

Sample & Assay Technologies miScript II RT Kit


Reverse transcription theory

Mix RNA, 5x miScript HiFlex or HiSpec Buffer, RNase-free water, 10x Nucleics Mix, and miScript

Reverse Transcriptase Mix Incubate at 37 °C for 60 min Incubate at 95 °C for 5 min

Use the cDNA to set up real-time PCR

reactions

miScript II Reverse Transcription Procedure


Sample & Assay Technologies miScript II RT: Exceptional Linearity


Linear over 6 logs of input RNA

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miR-16

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Linear (miR-20a)

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miR-16

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Sample & Assay Technologies miScript II PCR: Exceptional Sensitivity


Detection of 10 copies to >10 6 copies of miRNA


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Log copy number of miRNA using the HiSpec Buffer

Mean CT

miR-21

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Log copy number of miRNA using the HiFlex Buffer

Mean CT

miR-21

Linear (miR-21)

Sample & Assay Technologies miScript PCR System: Exceptional Specificity


Excellent discrimination between closely related miRNA family members

Relative detection (as % of perfect match)

cDNA used in PCR

miScript Primer Assay Used

Let-7b Let-7c miR-98 Let-7d Let-7e Let-7a Let-7f Let-7g Let-7i

Let-7b 100.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Let-7c 0.5 100.0 0.0 0.0 1.0 0.1 0.0 0.0 0.0

miR-98 0.0 0.2 100.0 0.1 0.0 0.1 0.0 0.0 0.1

Let-7d 0.1 0.0 0.0 100.0 0.0 0.4 0.0 0.0 0.0

Let-7e 0.1 0.0 0.0 0.0 100.0 0.2 0.0 0.0 0.0

Let-7a 0.1 0.6 0.0 0.5 3.9 100.0 0.1 0.0 0.0

Let-7f 0.6 0.1 0.0 0.1 0.0 1.1 100.0 0.1 0.1

Let-7g 0.6 0.2 0.0 0.1 0.0 0.0 0.0 100.0 0.2

Let-7i 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 100.0

HiFlex Buffer



miRNA Profiling Technologies

Functionalization

Isolation


Sample & Assay Technologies miRNA expression profiling applications


� Mechanisms of gene regulation

� Developmental biology

� Novel miRNA discovery

� Studying miRNA–mRNA and miRNA–protein interactions

� Integrative analyses of miRNAs in the context of gene regulatory networks

� Biomarkers

� Tissue-based miRNA biomarkers

� Tissues of unknown origin

� Circulating biomarkers

� Forensics

From Pritchard, C.C., et al, Nature Rev. Genet 2012, 13, 358-369

Sample & Assay Technologies miRNome miScript miRNA PCR Arrays


Leading coverage and validated assays

� Human� Mouse� Rat � Dog� Rhesus macaque

� 100% validated assays� Each assay is bench validated� Each array is quality controlled

� Leading miRNome coverage

� Customizable

� miRBase V17 and V18 assays are available!

� Contact us if you are interest in a different species!

miRNome Arrays Benefits of miRNome Arrays

Species Assays(miRBase V16)

Human 1066

Mouse 940

Rat 653

Dog 277

Rhesus macaque 469 (V18)

Sample & Assay Technologies Focused miScript miRNA PCR Arrays


Biologically relevant, intelligently designed

Focused Arrays

� miFinder� Cancer PathwayFinder� Brain Cancer � Breast Cancer� Ovarian Cancer� Liver miFinder – New!� Apoptosis� Cell Differentiation & Development� Immunopathology � Inflammatory Response & Autoimmunity� Diabetes � Neurological Development & Disease � T-Cell & B-Cell Activation� Prostate Cancer� Cardiovascular Disease� Serum & Plasma

� 100% validated assays� Each assay is bench validated� Each array is quality controlled

� Biological relevant and up-to-date

� Customizable

� Contact us if you are interest in a different species!

Benefits of Focused Arrays

Sample & Assay Technologies High Content (HC) miScript miRNA PCR Arrays


Targeted miRNome profiling

� miFinder 384HC

� Serum & Plasma 384HC

� Cancer PathwayFinder 384HC

� Liver miFinder 384HC – New!

Sample & Assay Technologies Anatomy of a miScript miRNA PCR Array


96-well Format: 84 miRNA + 12 controls

Cel-miR-39

miScript PCR Controls for Normalization

miRTC PPCSNORD61; SNORD68; SNORD72 SNORD95; SNORD96A; RNU6-2

RTControl

PCRControl

Spike in Control

84 miRNAs

� Cel-miR-39� Alternative data normalization using exogenously spiked Syn-cel-miR-39 miScript miRNA Mimic

� miScript PCR Controls� Data normalization using the ∆∆CT method of relative quantification

� miRNA reverse-transcription control (miRTC)� Assessment of reverse transcription performance

� Positive PCR control (PPC)� Assessment of PCR performance

Sample & Assay Technologies miScript miRNA PCR Arrays


Formats built to fit your experiment and cycler

96-well 384-well

384-well (4 x 96) Rotor-Disc 100

Sample & Assay Technologies miScript miRNA PCR Arrays


Compatible with a wide range of instruments

� 96-Well: 7000, 7300, 7500, 7700, 7900HT, ViiA 7� FAST 96-Well: 7500, 7900HT, Step One Plus, ViiA 7� FAST 384-Well: 7900HT, ViiA 7

� Mastercycler ep realplex 2/2S/4/4S

� Mx3000p, Mx3005p, Mx4000p

� iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384� Opticon, Opticon 2, Chromo 4

� LightCycler 480

� TP-800

RotorGene Q



QIAGEN PCR Array Service Core

miScript miRNA PCR Arrays

� Total RNA Isolation: miRNeasy Kits

� Reverse Transcription: miScript II RT Kit

� qPCR: miScript miRNA PCR Arrays

� Data analysis included!

Send your samples and receive results!



Rapid Workflow = Robust, Reproducible Performance


y = 1.0075x + 0.2891

R2 = 0.989

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Mean CT: Biological Replicate 1

Mean CT: Biological Replicate 2

1st Time Array User

� Total HeLa S3 (miRNeasy)� Pellet 1: Frozen June 2010� Pellet 2: Frozen April 2011

� HiSpec Buffered cDNA� miScript real-time PCR

� miFinder miScript miRNA PCR Array

� 1 hour� HiSpec Buffer

� 2 minutes

� 2 hours

� 15 minutes



Rapid Workflow = Robust, Reproducible Performance


y = 0.9994x - 0.1945

R2 = 0.9959

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CT: Operator 1CT: Operator 2

Two Operators

� Total HeLa S3 (miRNeasy)� HiSpec Buffered cDNA� miScript real-time PCR

� miFinder miScript miRNA PCR Array� Rotor-Gene Q

� 1 hour� HiSpec Buffer

� 2 minutes

� 2 hours

� 15 minutes

Sample & Assay Technologies Focused miRNA expression profiling


FFPE samples

� One 5 µm FFPE section used per FFPE isolation� Each isolation is from a different section� On average, each isolation provided enough total RNA for:

– Two full human miRNome profiles– Ten pathway-focused PCR arrays

� RT: 125 ng total RNA, HiSpec Buffer� qPCR: Human miFinder miScript miRNA PCR Array (0.5 ng cDNA per well)

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Normal Lung

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Lung Tumor

Sample & Assay Technologies Focused miRNA expression profiling (cont.)


FFPE samples

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2-∆CT

: Normal Lung FFPE Tissue

2-∆CT: Tumor Lung FFPE Tissue

2-∆CT: Tumor vs. Normal

� Significant differences exist between the mature miRNA expression levels of the two tissue types

� ± 2-fold [red lines] used as a cutoff for significance

Sample & Assay Technologies High Content (HC) miRNA expression profiling


Serum sample

2-∆CT: Cancer vs. Normal

� Workflow� 200 µl serum � 14 µl total RNA � 1.5 µl total RNA, HiSpec Buffer � Serum & Plasma 384HC miScript

miRNA PCR Array

� Significant differences exist between the mature miRNA expression levels of the two tissue types

� ± 3-fold [red lines] used as a cutoff for significance

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: Normal Serum

2-∆CT: Lung Cancer Serum

Fold-Regulation: Cancer vs. Normal

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Sample & Assay Technologies miRNome miRNA expression profiling


HCT 116 colorectal cancer cells

Scatter Plot Volcano Plot

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: Untreated

2-∆CT: Treated

� 5-aza-2’-dC irreversibly inhibits DNA methyltransferase driven DNA methylation reactions by incorporating into DNA and covalently binding to the active site of the DNMT.

� Scatter Plot: Significant differences exist in the mature miRNA expression levels of the two samples that were tested

� 104 miRNAs were strongly up-regulated in 5-aza-2’-dC treated cells, while 30 were strongly down-regulated in 5-aza-2’-dC treated HCT 116 cells.

� Volcano Plot: When a p Value of 0.05 is applied, the expression up-regulation of 89 of the 104 miRNAs is significant, and the expression down-regulation of 21 of the 30 miRNAs is significant

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Log2 (Fold-Regulation)

p Value



Limiting Samples

Functionalization

Isolation


Body fluidsFFPE samplesLCM samplesFlow-sorted cellsCirculating tumor cellsFine needle biopsies

Sample & Assay Technologies miScript PreAMP PCR Kit


Straightforward, robust, and reproducible

y = 0.9973x + 0.2058

R2 = 0.9913

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Mean CT: Operator 1Mean CT: Operator 2

� Universal reference RNA� Two operators

� Experienced operator� First time operator

� miScript real-time PCR� miFinder miScript miRNA PCR Array

Sample & Assay Technologies miScript PreAMP: Excellent result, a fraction of th e input


Limiting FFPE sample

y = 0.9934x - 0.1457

R2 = 0.9606

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∆CT: PreAMP

� Workflow� No PreAMP: 250 ng total RNA, HiSpec Buffer � Human miRNome miScript miRNA PCR Array (Plate 1)� PreAMP: 10 ng total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP � preamplified cDNA

used for Human miRNome miScript miRNA PCR Array (Plate 1)

� Excellent correlation, PreAMP vs. No PreAMP� Preamplified cDNA provides enough material for more than 2 full human miScript

miRNome profiles. Input was only 1 ng of cDNA!

Sample & Assay Technologies miScript PreAMP: Excellent result, a fraction of th e input


Limiting serum sample

1.E-01

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2-∆CT

: Normal Serum

2-∆CT: Colorectal Cancer Serum

� Workflow� 5 µl serum � 14 µl total RNA � 2 µl total RNA, HiSpec Buffer � one-tenth of cDNA, miScript PreAMP �

preamplified cDNA (0.07 µl SE) used for Human Serum & Plasma 384HC miScript miRNA PCR Array

� Conclusion: Starting with only 5 µl of serum, significant differences were detected when colorectal cancer serum was compared to normal serum

y = 0.9728x + 0.7671

R2 = 0.9757

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CT: Individual RNA Isolation from Serum

CT Mean: 3 RNA Isolations from Serum

Reproducibility 2 -∆CT: Colorectal Cancer vs. Normal

Sample & Assay Technologies Let miScript drive your miRNA studies!


� 1 hour

� 2 minutes

� 2 hours

� 15 minutes

Sample

Results!



miRNA Functionalization Technologies

Functionalization

Isolation


Sample & Assay Technologies Manipulating miRNA function


Sample & Assay Technologies miScript miRNA Mimics


Mock endogenous miRNA activity

Features of miScript miRNA Mimics:� Double-stranded RNA oligonucleotide� Functional sequence is the same as the natural mature miRNA� Transfection results in inhibition comparable to the endogenous miRNA� Stable in culture for up to 72 hours

Sample & Assay Technologies miScript miRNA Inhibitors


Block endogenous miRNA activity

Features of miScript miRNA Inhibitors:� Single-stranded, chemically modified RNA oligonucleotide� Designed and modified to ensure efficient inhibition of endogenous miRNA� Transfection of inhibitor counteracts miRNA-induced silencing � Effective for at least 96 hours following transfection

Sample & Assay Technologies miScript miRNA Mimics and Inhibitors


Controls

Mimic negative control

AllStars Negative Control siRNA� No homology to any mammalian gene.� Validated for non-effects in microarray and phenotype assays.� Validated for RISC entry

Mimic positive control

Syn-hsa-miR-1 mimic� Not expressed under most culture conditions, only expressed in muscle cells � To check for optimal conditions by using miScript PCR miR-1

Inhibitor negative control

miScript Inhibitor Negative Control� Target the sequence of miScript mimic negative control

Inhibitor positive control

Anti-hsa-miR-1 inhibitor � To check for optimal conditions in combination with Syn-hsa-miR-1 mimic

Transfection control

AllStars Hs Cell Death Control siRNA� Cell death = successful transfection� To run in every experiment

Sample & Assay Technologies RT2 Profiler miRNA Targets PCR Array


What genes are regulated by your miRNA of interest?

Benefits:� Assist miRNA functional analysis� Follow up on miRNA expression profiling analyses� Species currently covered: Human, mouse, and rat

Features:� Profiles expression of 84 verified & bioinformatically predicted target genes� Target genes specific for a miRNA & others in same seed sequence family� Free data analysis suite� Available in 2, 12 or 24 plate packs� Compatible with any real time qPCR instrument currently in your lab

Note: Uses RT 2 qPCR Reagent Kits

Sample & Assay Technologies Where can I find miScript miRNA PCR Arrays?


www.sabiosciences.com/mirna_pcr_array.php

� miRNA Overview

� miScript PCR System

� miScript miRNA PCR Arrays

� Products for functional studies

� miRNA purification options

Sample & Assay Technologies Where can I find miScript Primer Assays?


www.qiagen.com/GeneGlobe

Sample & Assay Technologies QIAGEN’s miRNA portfolio

58

Your miRNA workflow, from sample to results!

miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT K it and PreAMP Kit HiPerFect Transfection Reagent

miRNeasy 96 Kit miScript SYBR Green PCR Kit Attractene Transfection R eagent

miRNeasy FFPE Kit miScript miRNA PCR Arrays miScript miRNA Mimics

miRNeasy Serum/Plasma Kit miScript miRNA Data Analysi s Tool miScript miRNA Inhibitors

PAXgene Tissue miRNA Kit miScript Primer Assay Custom miScript miRNA Mimics

PAXgene Blood miRNA Kit miScript Precursor Assay miSc ript Target Protector

Supplementary protocol for miRNA from Plasma and Serum

miScript PCR Starter KitmiScript miRNA Inhibitor 96 and 384 Plates and Sets

Pro

filin

g

QIAcube QIAgility Rotor-Gene Q

Webinar 1: Meeting the Challenges of miRNA Research

QIAGEN Service Core

FunctionalizationIsolationQuantification and profiling

Sample & Assay Technologies Upcoming Webinars


Experimental Setup and miRNA Profiling Webinars

Webinar 3: Profiling miRNA expression: on the road to biomarker development


Webinar 2 : microRNA expression analysis: From experimental design to data analysis


Sample & Assay Technologies Thank you for attending


http://www.sabiosciences.com/promotion/miscriptdemo.php

Technical Support Contact Information

� Monday through Friday

� 8:00AM to 6:00PM EST

� Direct Phone: 888-503-3187

� E-Mail: [email protected]



Thank you for attending today’s webinar!

Jonathan Shaffer, Ph.D.

[email protected] Technologies, R&D Americas

Questions?

Documents

Mi rna part i_2013