Mi rna series i-dec 2012

Meeting the challenges of miRNA research:
microRNA Biogenesis, Function, and Analysis

Jonathan M. Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
microRNA 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

Welcome to the three-part webinar series on
miRNA and its role in human disease

Webinar 1 :
Date:
Speaker:

Webinar 2 :
Date:
Speaker:

Webinar 3 :
Date:
Speaker:

Meeting the challenges of miRNA research:
An introduction to microRNA biogenesis, function, and analysis
December 4, 2012
Jonathan Shaffer, Ph.D.

Advanced microRNA expression analysis:
From experimental design through data analysis
December 11, 2012

Profiling miRNA expression in Cells, FFPE, and serum:
On the road to biomarker development
December 18, 2012
Eric Lader, Ph.D.

-2-


Agenda

miRNA Background
miRNA Genomics
miRNA in Disease
miRNA Isolation Technologies
miRNA Quantification Technologies
miRNA Profiling Technologies
miRNA Functionalization Technologies
New product released!

-3-


RNA interference: A natural phenomenon
Discovery tool, potential diagnostic, potential therapeutic
25000

10000

Sequences (m iRBase)

20000

8000
7000

15000

6000
5000

10000

4000
3000

5000

2000

Refer ences (Highwir e + PubMed)

9000

1000
0

0

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Year

Virtually every publication includes characterization
by quantification.

May 26, 2003

Changes in miRNA can be correlated with gene
expression changes in development, differentiation,
signal transduction, infection, aging, and disease.
-4-


Canonical pathway of microRNA (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.

-5-


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
-6-


How to determine miRNA-mRNA interactions
Algorithms for predicting miRNA-mRNA interaction

Target Prediction is based on:
Bioinformatics

.

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

.

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

Wet lab research
Empirical evidence from microarrays
Reporter systems
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/
-7-


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
-8-


Isolation

Quantification

Functionalization

miRNA Genomics
Generating Diversity in the miRNome

-9-


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

- 10 -


Multiple loci can generate the same mature miRNA
Biogenesis creates incredible diversity
Stem Loop

CHR

Overlapping transcripts

CHR: Coordinates (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

1302-6

7

intronic

Protein coding/HDAC9; intron 1

7: 18166843-18166932 [-] ; Antisense

1302-8

9

intronic

Protein coding/ch9orf174

9: 100125836-100125963 [-]; Antisense

20: 49231173-49231322 [-]; Sense

Mature-miR-1302: UUGGGACAUACUUAUGCUAAA

www.mirbase.org
- 11 -


Post-transcriptional modification of miRNA
Generation of miRNA diversity by processing and ADAR editing
ADAR: adenosine deaminase, RNA specific
A-> I destabilizes dsRNA

Deep sequencing of hsa-miR-21-5p

A I

ADAR

Pri-miRNA
DROSHA Processing
Block

A I

Pre-miRNA
Exportin

ADAR

ADAR

www.mirbase.org

- 12 -

A I

A I

DICER
Processing
Block
Change in
miRNA seed

Kawahara, et. al (2007) Science. 315 (5815):1137-40

Isolation

Quantification

Functionalization

miRNA in Disease

- 13 -


Potential events that disrupt normal miRNA activity
Disruption of miRNA-mRNA interaction
Altered Transcription

microRNA Gene
Pri-miRNA

Methylation
Histone Modification

Genomic Instability
Amplification/Deletion
Translocation

Drosha-DGCR8

Transcription Factor

Insertional Mutagenesis

Pre-miRNA

Drosha Processing
Exportin

DICER-TRBP

Dicer Processing
mature miRNA

Ago

Loss of miRNA
Binding Site in target
SNP or Mutation

miRNP

Alternative Splicing
Target Transcript
- 14 -

Loss/Change of 3’-UTR

A look back 10 years
Unique miRNA signatures are found 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 cancers (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 studies and reviews have
associated the presence of various miRNAs with cell proliferation,
resistance to apoptosis, invasiveness, and differentiation in cancer cells.

- 15 -


Isolation

Quantification

Functionalization

miRNA Isolation Technologies

- 16 -


miRNeasy Kits: Mini, Micro
Flexible protocols: total RNA or enrichment of miRNA

High quality, pure RNA

.

Suitable for sensitive downstream
applications

Rapid procedure

.

Streamlined protocol for lowthroughput 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

- 17 -


miRNeasy FFPE Kit
Enables purification of 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

- 18 -


miRNeasy Serum/Plasma Kit
Industry standard: Purification of miRNA from serum and plasma

For purification of circulating RNA from
animal and human plasma and serum
Includes synthetic RNA control assay for normalization

Clarified plasma
or serum

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

Plasma

QIAzol

Bind

Serum

Wash

Elute

Manual or Automated on QIAcube

Automatable protocol

- 19 -


Isolation

Quantification

Functionalization

miRNA Quantification
Technologies

- 20 -


Next generation miScript PCR System
miRNA Profiling Redefined

1.

miScript II RT Kit
HiFlex Buffer: Unparalleled flexibility for quantify 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: New!
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 MM
Universal Primer

6.

miScript miRNA PCR Array data analysis software
Straightforward, free data analysis

- 21 -


miScript PCR System
miRNA Profiling and Quantification Re-defined

miScript II RT
HiFlex Buffer

HiSpec Buffer

What it allows

What it allows
Specific detection of Mature miRNAs

Flexible detection of all RNA molecules
- Mature miRNAs
- Precursor miRNAs
- mRNAs
- Other non coding RNAs

When to use it

When to use it

When mRNAs/precursors/other long
non coding RNAs quantified in parallel
with mature miRNAs

Only mature miRNA detection is desired
- Single miScript Primer Assays
- miScript miRNA PCR Arrays
- miRNome miScript miRNA PCR Arrays

HiSpec chemistry is analogous to the RT2 miRNA
First Strand cDNA Synthesis Kit chemistry
- 22 -


When should you use HiFlex Buffer or HiSpec Buffer?

HiFlex Buffer

HiSpec Buffer

Should be used to prepare cDNA for
the quantification of both mature
microRNAs & mRNAs using
appropriate primer assays.

Should be used to prepare cDNA for
mature miRNA quantification. Long
RNAs, such as mRNAs, are not
converted into cDNA. HiSpec is the
optimized and exclusive buffer for
mature miRNA profiling with miScript
miRNA PCR Arrays.

30

25

25
Mean CT

35

30

20
15

20
15

- 23 -

miR-25

miR-21

miR-16

Let-7a

MAPK1

CDK1

PPIA

GAPDH

miR-25

miR-21

0
miR-16

0
Let-7a

5
MAPK1

5
CDK1

10

PPIA

10

GAPDH

M ean C T

35


miScript PCR System
Reverse Transcription & PCR
HiFlex Buffer

HiSpec Buffer

miScript II Reverse Transcription Procedure
Mix RNA, 5x miScript HiFlex or
HiSpec Buffer, RNase-free water,
10x Nucleics Mix, and miScript
Reverse Transcriptase Mix

Use the cDNA to set
up real-time PCR
reactions

Incubate at 37°C for 60 min

- 24 -

Incubate at 95°C for 5 min


miScript II RT: Exceptional Linearity
Linear over 6 logs of input RNA

HiFlex Buffer

HiSpec Buffer

40

40

miR-16

miR-16
miR-20a

35

Linear (miR-20a)

miR-21
Linear (miR-16)
Linear (miR-20a)

30

Linear (miR-21)

Mean CT

Mean CT

30

miR-20a

35

miR-21
Linear (miR-16)

25

Linear (miR-21)

25

20

20

15

15

10

10
-2

-1

0

1

2

3

4

-2

Log (ng) of RNA in cDNA synthesis using the HiFlex Buffer

-1

0

1

2

3

4

Log (ng) of RNA in cDNA synthesis using the HiSpec Buffer

- 25 -


miScript II PCR: Exceptional Sensitivity
Detection of 10 copies to >106 copies of miRNA. e.g. miR-21

HiFlex Buffer

HiSpec Buffer
32

32

miR-21
Linear (miR-21)

miR-21
Linear (miR-21)

28

Mean CT

Mean CT

28

24

24

20

20

16

16

12

12
1

2

3

4

5

6

1

Log copy num ber of m iRNA using the HiFlex Buffer

2

3

4

5

6

Log copy num ber of miRNA using the HiSpec Buffer

- 26 -


miScript PCR System: Exceptional Specificity
Excellent discrimination between Let-7 family isoforms

HiFlex Buffer
Relative detection (as % of perfect match)
miScript Primer Assay Used

cDNA used
in PCR

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

- 27 -


Isolation

Quantification

Functionalization

miRNA Profiling
Technologies

- 28 -


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
- 29 -


miRNome miScript miRNA PCR Arrays
Redefining miRNA expression profiling

miRNome Arrays

Benefits of miRNome Arrays

Human
Mouse
Rat
Dog
Rhesus macaque
Species

100% validated assays
Each assay is bench validated
Each array is quality controlled
Leading miRNome coverage

Assays
(miRBase V16)

Human
Mouse

940

Rat

653

Dog

277

Rhesus macaque

Customizable

1066

469 (V18)

miRBase V17 and V18 assays are
available!
Contact us if you are interest in a
different species!

- 30 -


Focused miScript miRNA PCR Arrays
Redefining miRNA expression profiling

Benefits of Focused Arrays

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

- 31 -


New! High Content (HC) miScript miRNA PCR Arrays
An economical alternative to whole miRNome expression profiling
miFinder 384HC
Profiles the expression of the 372 most abundantly expressed and best characterized
miRNAs in miRBase.

Serum & Plasma 384HC
Profiles the expression of 372 miRNAs detectable in serum and plasma using the
miScript PCR System. Content is derived from in-house miRNome (miRBase V18)
profiling of more than 100 normal and disease serum and plasma samples.

New! Cancer PathwayFinder 384HC

- 32 -


Pathway-Focused: 84 miRNAs + 12 Controls

84 miRNAs

Cel-miR-39

SNORD61; SNORD68; SNORD72
SNORD95; SNORD96A; RNU6-2

miRTC

PPC

Spike in
Control

miScript PCR Controls for
Normalization

RT
Control

PCR
Control

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
- 33 -


Available in 96-well, 384-well, and Rotor-Disc 100 Formats
96-well

384-well

384-well (4 x 96)

Rotor-Disc 100

- 34 -


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

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

Mastercycler ep realplex 2/2S/4/4S

LightCycler 480

Mx3000p, Mx3005p, Mx4000p

TP-800

RotorGene Q

- 35 -


QIAGEN PCR Array Service Core

Send your samples and receive results!

Total RNA Isolation: miRNeasy Kits
Reverse Transcription: miScript II RT Kit
qPCR: miScript miRNA PCR Arrays
Data analysis included!

- 36 -


Rapid Workflow = Robust and Reproducible Performance
1st Time Array User

2 minutes

30
Mean CT: Biological Replicate 2

1 hour
HiSpec Buffer

25

20

y = 1.0075x + 0.2891

2 hours

2

R = 0.989
15
15

20

25

30

Mean CT: Biological Replicate 1

15 minutes

- 37 -

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

Rapid Workflow = Robust and Reproducible Performance
Two Operators
1 hour
HiSpec Buffer

30

2 minutes

CT: O per ator 2

25

20

15

2 hours

y = 0.9994x - 0.1945
2

R = 0.9959
10
10

15

20

25

30

CT: O per ator 1

15 minutes

- 38 -

Total HeLa S3 (miRNeasy)
HiSpec Buffered cDNA
Rotor-Gene Q

miRNA expression profiling using FFPE samples
Normal Lung

40

36

32

32

28

28

24

24

CT Value

36

CT Value

Lung Tumor

40

20

20
16

16
12

12

FFPE Isolation 1
FFPE Isolation 2

8

FFPE Isolation 1
FFPE Isolation 2

8

FFPE Isolation 3

FFPE Isolation 3

4

4
1

1

7 13 19 25 31 37 43 49 55 61 67 73

7 13 19 25 31 37 43 49 55 61 67 73

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)
- 39 -


miRNA expression profiling using FFPE samples (cont.)
2-∆CT: Tumor vs. Normal

- ∆CT

: Tumor Lung FFPE Tissue

1.E+ 04
1.E+ 03
1.E+ 02
1.E+ 01
1.E+ 00
1.E-01
1.E-02

2

1.E-03
1.E-04
1.E+ 04

1.E+ 03

1.E+ 02

1.E+ 01

1.E+ 00

1.E-01

1.E-02

1.E-03

1.E-04

2

- ∆CT

: Nor m al Lung FFPE Tissue

Significant differences exist between the mature miRNA
expression levels of the two tissue types
± 2-fold [red lines] used as a cutoff for significance

- 40 -


miRNA expression profiling using serum samples
Serum & Plasma 384HC
2-∆CT: Cancer vs. Normal

Fold-Regulation: Cancer vs. Normal
20
Fold-Regulation: Cancer vs. Nor m al

1.E+ 00

1.E-01

1.E-02

1.E-03

2

- ∆CT

: Lung Cancer Ser um

1.E+ 01

1.E-04
1.E+ 01

1.E+ 00

1.E-01

1.E-02

1.E-03

1.E-04

2

15

10

5

0

-5

-10

- ∆CT

: Nor mal Ser um

Workflow
200 µl serum
14 µl total RNA
miRNA PCR Array

1.5 µl total RNA, HiSpec Buffer

Serum & Plasma 384HC miScript

Significant differences exist between the mature miRNA expression levels of the two tissue
types
± 3-fold [red lines] used as a cutoff for significance

- 41 -


miRNome expression profiling using HCT 116 cells
Scatter Plot

Volcano Plot

1.E+ 02

0.00001

0.0001

1.E+ 00
1.E-01

p Value

2

- ∆CT

: Tr eated

1.E+ 01

1.E-02
1.E-03

0.001

0.01

1.E-04
0.1

1.E-05
1.E+ 02

1.E+ 01

1.E+ 00

1.E-01

1.E-02

1.E-03

1.E-04

1.E-05

2

1
-8

- ∆CT

: Untr eated

-6

-4

-2

0

2

4

6

8

Log 2 (Fold-Regulation)

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 downregulated 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
- 42 -


Isolation

Quantification

Functionalization

Limiting Samples
Body fluids
FFPE samples
LCM samples
Flow-sorted cells
Circulating tumor cells
Fine needle biopsies

- 43 -


miScript PreAMP PCR Kit
Straightforward protocol, reproducible and robust amplification

Mean CT: O per ator 2

30

25

20
y = 0.9973x + 0.2058
2

R = 0.9913
15
15

20

25

30

Mean CT: O per ator 1

Universal reference RNA
Two operators
Experienced operator
First time operator

- 44 -


miScript PreAMP: miRNA expression profiling from limiting FFPE samples
Excellent results with a fraction of the input
15
13
11

∆CT: PreAMP

9
7
5
3
1

-1
y = 0.9934x - 0.1457
-3

2

R = 0.9606

-5
-5

-3

-1

1

3

5

7

9

11

13

15

∆CT: No Pr eAMP

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!
- 45 -


miRNA expression profiling using limited serum samples
Using the Serum & Plasma 384HC
2-∆CT: Colorectal Cancer vs. Normal

Reproducibility

: Color ectal Cancer Ser um

1.E+ 03

28

24

y = 0.9728x + 0.7671
2

R = 0.9757

CT: Individual RNA Isolation fr om Ser um

2

1.E+ 03

32

1.E+ 02

28

1.E+ 01

24

1.E+ 00

1.E-01

20

1.E+ 00

1.E-01

16
16

1.E+ 01

- ∆CT

20

1.E+ 02

2

CT Mean: 3 RNA Isolations fr om Ser um

32

- ∆CT

: Nor m al Ser um

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
- 46 -


Let miScript drive your miRNA expression profiling!

Sample
1 hour

2 minutes

2 hours

Results!

15 minutes

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Isolation

Quantification

Functionalization

miRNA Functionalization
Technologies

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Manipulating miRNA function

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miScript miRNA Mimics act just like endogenous miRNA

.

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

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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

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miScript miRNA Mimic & Inhibitor 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

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New product! RT2 Profiler miRNA Targets PCR Array
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 RT2 qPCR Reagent Kits
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53

www.sabiosciences.com/mirna_pcr_array.php

miRNA Overview
miScript PCR System
Products for functional studies
miRNA purification options
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www.qiagen.com/GeneGlobe
Single Primer Assays

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Your microRNA workflow, from sample to results!

miRNeasy FFPE Kit
miRNeasy Serum / Plasma Kit

miScript II RT Kit and PreAMP Kit

HiPerFect Transfection Reagent

miScript SYBR Green PCR Kit

Attractene Transfection Reagent

miScript miRNA PCR Arrays *

miScript miRNA Mimics

miScript miRNA Data Analysis Tool

miRNeasy 96 Kit

Profiling

miRNeasy Mini Kit, miRNeasy Micro Kit

miScript miRNA Inhibitors

PAXgene Tissue miRNA Kit

miScript Primer Assay

Custom miScript miRNA Mimics

PAXgene Blood miRNA Kit

miScript Precursor Assay

miScript Target Protector

Supplementary protocol for miRNA from
Plasma and Serum

miScript PCR Starter Kit

miScript miRNA Inhibitor 96 and 384
Plates and Sets

* System is compatible with any real-time instrument

QIAcube

QIAgility
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Rotor-Gene Q

Upcoming webinars
Webinar 2 : microRNA expression analysis:
From experimental design
to data analysis
Date:

December 11, 2012, 9:30 am EST

Speaker:


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

December 18, 2012, 9:30 am EST

Speaker:

Eric Lader, Ph.D.

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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Free or Discounted miScript miRNA PCR Arrays!

Technical Support Contact Information
M-F, 8am to 6pm EST (US)
Direct phone: 888-503-3187
Email: support@sabiosciences.com

Let miScript drive your miRNA studies!

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Questions?

Thank you!

Jonathan Shaffer
Jonathan.Shaffer@qiagen.com

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
- 59 -


Mi rna series i-dec 2012

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