Mi rna assays 2012

From Cellular to Molecular –
New Assays for miRNA Research
Samuel J. Rulli, Jr. Ph.D.
Samuel.Rulli@Qiagen.com

Sample & Assay Technologies

From Cellular to Molecular –
New Assays for miRNA Research
Samuel J. Rulli, Jr. Ph.D.
Samuel.Rulli@Qiagen.com

Questions, Comments, Concerns?
US Applications Support
888-503-3187
̣

Global Applications Support
SABio@qiagen.com

support@sabiosciences.com


miRNA Analysis: From Cellular to Molecular

Isolation
miRNeasy

miRNA
Studies

Target Identification

Expression
miScript miRNA PCR Arrays

SureFIND Transcriptome PCR Arrays

Function
miRNA mimics & inhibitors
Cignal Reporters

-3-


What Is Micro RNA?

RISC

Target mRNA

5’
CDS

3’

3’

5’
miRNA

Seed region

Endogenously expressed small functional RNAs (~ 21nt -24 nt)
Regulate mRNA expression post-transcriptionally
Involves the formation of RISC (RNA induced Silencing Complex)
Translational Repression and or mRNA degradation

Only partial sequence complementarity required
A given miRNA can regulate multiple mRNAs
A given mRNA can be regulated by multiple miRNAs
Another layer of complexity for regulating gene expression

Aberrant expression of miRNAs is associated with many diseases
e.g. Many different Cancers

-4-


Canonical pathway of microRNA 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-


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

-6-


Multiple loci can generate the same mature miRNA
But are under different regulatory control
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; intron4

1302-6

7

intronic

Protein coding/HDAC9; intron 1

1302-8

9

intronic

Protein coding/ch9orf174

20: 49231173-49231322 [-]; Sense
7: 18166843-18166932 [-] ; Antisense
9: 100125836-100125963 [-];
Antisense

Mature-miR-1302: UUGGGACAUACUUAUGCUAAA

www.mirbase.org

-7-


A growing picture of miRNA dysregulation in cancer pathways

In the last 5 years, a substantial number of studies have associated
various miRNAs with cell proliferation, resistance to apoptosis, differentiation,
immunity and cancer initiation, progression and metastasis.
.

.

For example:

.

Deletions of miRNA-regulated genes have been detected in more than 65%
of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in
16% to 40% of multiple myelomas, and in 60% of prostate cancers.
Other miRNA abnormalities have been reported in a wide variety of
Human neoplasms, including other hematologic malignancies such as
promyelocytic leukemia
Benign tumors such as leiomyoma and pituitary adenoma
Multiple types of carcinomas, including pancreatic, esophageal,
thyroid, lung, and breast
Neuroblastomas and glioblastomas

-8-


Why Analyze miRNA?
Easy to profile entire miRNome with only ~1000 miRNAs

.

Complexity of miRNA/mRNA interaction makes them exciting and
unknown epigenetic mechanism of gene regulation
.

miRNA regulated genes are involved in a variety of biological
processes
Cancer
Development
Immunology
Aging
Heart Diseases
Neurological Diseases
Diabetes
.

miRNAs are promising biomarkers for early disease detection and prognosis
-9-



Isolation
miRNeasy

miRNA
Studies


Expression
and PCR Assays


Function
Cignal Reporters

- 10 -


miRNeasy
Purification of miRNAs

miRNA purification
Cells and tissues

miRNeasy Mini Kit

Cells and tissues (small amounts)

miRNeasy Micro Kit

Cells and tissues, 96-well format

miRNeasy 96 Kit

Formalin-fixed, paraffin-embedded tissues

miRNeasy FFPE Kit

Plasma and serum

miRNeasy Serum/Plasma Kit

Almost any sample source can yield high quality miRNA for miRNA profiling

- 11 -


miRNA isolation is simple from any sample

- 12 -



Sufficient for miScript
miRNA PCR Arrays

- 13 -



Maximize limited
sample amounts to
measure miRNA
and mRNA by PCR
arrays or individual
qPCR Assays

- 14 -



Isolation
miRNeasy

miRNA
Studies


Expression
and PCR Assays


Function
Cignal Reporters

- 15 -


How to Monitor miRNA Expression Levels

Northern Blot
Very low throughput
Very time consuming and complex
Specificity issue
Microarrays
Profile More Sequences
Poor Sensitivity & Dynamic Range, Complicated Protocol
Expensive equipment
Real-Time RT-PCR (qPCR)
Simpler Protocol
Better Sensitivity, Specificity & Reproducibility
No investment in equipment
Profile up to 384 miRNA in a single run (miRNome needs 3x 384 plates)

.

.

.

- 16 -


miScript miRNA PCR Array System
miRNeasy miRNA Isolation Kit

.

miScript miRNA 1st Strand cDNA Synthesis Kit
Preferentially reverse transcribe mature miRNA
Built-in external RNA control
One-step reaction
miScript miRNA PCR Arrays and qPCR Primers
Human, mouse, rat: Search miRNA Primers:
http://www.sabiosciences.com/mirna_pcr_assay.php

.

.

-or-

PCR Arrays: Pathways and Genomes
Cancer
Cell Differentiation & Development
Immunopathology
Inflammation
miFinder
Whole Genome (miRNome) – Sanger miRBase V14.0
Serum (or plasma)
Brain Cancer miRNA
Neurological Development & Disease
Optimized miScript SYBR® Green Master Mix
miScript miRNA Data Analysis Excel Template & Web Portal

.

.

- 17 -


cDNA Synthesis (miScript II RT KIT)
1 hours

.

Load Plates (Preferably with 8-Channel
Pipettors)
2 minutes

.

Run 40 cycle qPCR Program
2 hours

.

Upload and Analyze Data
15 minutes

.

- 18 -


Universal miRNA RT-PCR used by
miScript miRNA PCR Arrays and Assays

Same cDNA preparation can assay ANY miRNA now or in the future (think miRBase 24)

miRNA
Poly(A) Polymerase

AAAAAAAA
NNTTTTTTTT

Universal RT Primer site

Reverse Transcriptase

TTTTTTTT
Real-Time PCR

miRNA-Specific Primer

TTTTTTTT
Universal Primer
- 19 -


Performance: Universal RT Advantages
Benefit of universal RT 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 for future discveries

- 20 -


1 hours

.

Pipettors)
2 minutes

.

2 hours

.

15 minutes

.

- 21 -


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
- 22 -


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

384-well

384-well (4 x 96)

Rotor-Disc 100

- 23 -


Redefining miRNA expression profiling

Complete miRNome Arrays

Pathway and Disease -Focused Arrays
Human, Mouse, Rat:
miFinder (Dog and Rhesus macaque)
miFinder 384 HC (high content)

Human
Mouse
Rat
Dog
New! Rhesus macaque
Species

Cancer PathwayFinder
Brain Cancer
Breast Cancer
Cancer PathwayFinder
Ovarian Cancer
Prostate Cancer

Assays

Immunopathology
T cell & B cell Activation
Inflammatory Response & Autoimmunity
Neurological Development & Disease

(miRBase V16)

Human

1066

Mouse

940

Rat

653

Dog

277

Rhesus macaque

469 (V18)

Cell Differentiation & Development (Stem
Cell)
Serum & Plasma
Fully customizable!

- 24 -


1 hours

.

Pipettors)
2 minutes

.

2 hours

.

15 minutes

.

- 25 -


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

- 26 -


1 hours

.

Pipettors)
2 minutes

.

2 hours

.

15 minutes

.

- 27 -


Web Portal Location

http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php
- 28 -


Analyze Results

Average ∆Ct

Fold Change

- 29 -

Fold Regulation


Visualization of Data

Heat Map

- 30 -



Scatter Plot

- 31 -



Volcano Plot

- 32 -



Clustergram

Clustergram

- 33 -


Application Examples

Cancer
Identify miRNA biomarkers
Development & Differentiation
Regulation of Cell Type by miRNAs
Genome-Wide Discovery
Profile downstream miRNA expression

- 34 -


Application Data:
Which Cancer-related miRNAs are expressed in a specific tumor?

Human miScript CancerFinder miRNA PCR Array:
Identified miRNA dysregulated in a Colon Tumor.

- 35 -


Application Data: Cell Differentiation & Development

26 Brain-Specific miRNA

20 Muscle-Specific miRNA

Human Cell Differentiation & Development miScript miRNA PCR
Array: Identifies 26 Brain- and 20 Muscle-Specific miRNAs

- 36 -


Application Data: Genome-Wide Screening
40

Ct Adeno-p53

35

30

203
551a
25

34a
940

20

15
15

20

25

30

35

40

Ct Control

Human miScript miRNome PCR Array: Identifies Known and
Novel miRNA Targets of the p53 Signaling Pathway
- 37 -


miRNA expression profiling using FFPE samples
40

One 5 µM FFPE section used per FFPE isolation

RT: 125 ng total RNA, HiSpec Buffer
qPCR: Human miFinder miScript miRNA PCR Array
–
0.5 ng cDNA per well, Human miFinder PCR Array
∆CT (miRTC – PPC) is used to assess reverse transcription
efficiency

32
28
CT Value

RT and qPCR

Normal Lung

36

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

24
20
16
12

FFPE Isolation 1
FFPE Isolation 2

8

FFPE Isolation 3

20

∆CT < 7 indicates no inhibition
of reverse-transcription

18
16

1
40

32

10

28
CT Value

12

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

Lung Tumor

36

14

8
6

24
20

63
T:

61
T:

52
T:

43
T:

T:

41

32
T:

T:

T:

T:

N
:2

N
:1

23

8
21

0
12

12

-2

2

-3

16

-1

4

N
:1

∆CT (miRTC - PPC)

4

FFPE Isolation 1
FFPE Isolation 2
FFPE Isolation 3

4
1

Sample

7 13 19 25 31 37 43 49 55 61 67 73
Human miFinder miRNA Assay

- 38 -


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

Fold-Regulation: Tumor vs. Normal
14
Log2 Fold-Regulation (Tumor vs. Normal)

2

-∆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
1.E-03
1.E-04
1.E+04

1.E+03

1.E+02

1.E+01

1.E+00

-∆CT

1.E-01

1.E-02

1.E-03

1.E-04

2

: Normal Lung FFPE Tissue

10
6
2
-2
-6
-10
-14

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

- 39 -


miRNA expression profiling using serum samples
2-∆CT: Breast Cancer vs. Normal

Normal Serum

1.E+01
: Breast Cancer Serum

38

30
26

-∆CT

22
Serum Isolation 1

18

Serum Isolation 2
Serum Isolation 3

1.E-01
1.E-02
miR-34a

1.E-03
1.E-04
1.E-05
1.E+01

200 µl serum
50 µl total RNA 5 µl total RNA, HiSpec Buffer
miFinder miScript miRNA PCR Array

1.E+00

Workflow

-∆CT

1.E-01

2

1.E-02

miRNA

1.E-03

6 11 16 21 26 31 36 41 46 51 56 61

1.E-04

1.E-05

14
1

1.E+00

2

CT Value

34

: Normal Serum

one-half of cDNA used for 96-well

High reproducibility can be achieved using the miRNeasy Supplementary Protocol
Each isolation was from a different normal serum donor

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 -



Isolation
miRNeasy

miRNA
Studies


Expression
and PCR Assays


Function
Cignal Reporters

- 41 -


Cignal Reporter System: Measure Pathway Activity in Cells

Cignal Reporter Assays enable researchers to monitor transcription factor activation
Flexible system allows monitoring of different reporter assays
(GFP or Luciferase)
Available as single assays or grouped by biology or pathway
Validated assays for high signal to noise ratios

- 42 -


What Cignal Reporter Assays Provide
Functionally validated dual-reporter formulation
.

Minimizes variability, increasing the biological relevance of each
experiment

Ready-to-use reporters and controls, coupled with a transient reporter system
Enables simple and rapid analysis of the regulation of 45 signal
transduction pathways

Combination of custom-engineered transcriptional regulatory elements (TREs)
and destabilized, codon-optimized firefly luciferase
Increases the signal to noise ratio and maximizes assay sensitivity and
specificity

- 43 -


Cignal Reporter Assays: Complete Solution
How Cignal Reporter Assays Work
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box

Reporter Construct

firefly luciferase

Tandem repeats of

TRE

TF

FL

Upstream Signaling
Events
- 44 -


Reporter Design: Dual-Luciferase Assays
The Transcriptional Regulatory Elements
(TREs) are what establish the
pathway specificity of each reporter!
Pathway-targeted transcriptional
regulatory elements (45)

Reporter Construct

TRE

TATA
box

Firefly luciferase

Normalization Construct

CMV

TATA
box

Renilla luciferase

Constitutive transcriptional
regulatory element (1)

- 45 -


Cignal Reporter Assays (45 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X

Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR

- 46 -

Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
VDRE
Wnt
Xenobiotic

Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR


Cignal Pathway Reporter System Products
Primary cells, stem cells,
and difficult to transfect cell lines

Easy to
transfect cell lines

Endpoint
Assays

Cignal Dual-Luciferase
Reporter Assays

Single Pathway
Assays

10-Pathway
Arrays

Dynamic
Live Cell Assays

Dynamic
Live Cell Assays

Endpoint
Assays

Cignal GFP
Reporter Assays

Cignal Lenti
Luciferase Reporters

Single Pathway
Assays

Single Pathway
Assays

- 47 -

10-Pathway
Arrays

Cignal Lenti
GFP Reporters

Single Pathway
Assays


Cignal Reporters: What You Need

Cignal Reporter Assay
Reliable transfection reagent and
protocol (Attractene, HiPerFect)

Biological Test
miRNA mimics & Inhibitors
Detection Instrument and
Luciferase Substrates

- 48 -


Solutions for microRNA Function

QIAGEN miRNA Mimics and Inhibitors
For functional analysis of miRNA
QIAGEN Target Protectors
Disrupt Specific miRNA-Target Interactions

- 49 -


miRNA Mimics & Inhibitors: How do they work?

Transfection of
Mimics

Transfection of
Inhibitors

RISC

Target
Expression
Level

- 50 -



Transfection of
Mimics

Transfection of
Inhibitors

Mimics
increase
miRNA
activity

RISC

Target
Expression
Level

- 51 -



Transfection of
Mimics

Transfection of
Inhibitors

Inhibitor decrease
miRNA activity
RISC

Target
Expression
Level

- 52 -


Application Example: miRNA & Pathway Activity
The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression.

.

Expression of let-7 family members is downregulated in many cancer types and during tumor
progression in comparison to normal tissue.

.

Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and
miR-202)

.

Interestingly, certain let-7 species are also known to be upregulated in some cancer types.
These data on the expression of let-7 in various cancers indicate that individual let-7 family
members can have different activities.

.

Question: Which let-7 family members effect cell cycle progression and
how?

.

- 53 -


Cignal Reporters: What You Need

• Cignal Cell Cycle Pathway Reporter
Measures E2F/DP1 Activity

• Transfection of HEK293 cells

• Biological Test
•Let-7 family mimics

• Dual-Luciferase Assay

- 54 -


Let-7 Family Members and the Cell Cycle

- 55 -



Isolation
miRNeasy

miRNA
Studies


Expression
and PCR Assays


Function
Cignal Reporters

- 56 -


Discover miRNAs Regulating the Expression of any Gene


What regulates your gene?

miRNA

Upstream Pathway
Regulators

mRNA

GOI

DNA
Transcriptional
Factors
- 58 -


How do you identify those regulators?
Bioinformatics

Overexpression

.

.

Promoter Analysis

Chemical Treatments

.

.

×

mRNA

RNAi knockdown

Forward Genetics

.

.

×

DNA

- 59 -


Approach to Identifying miRNA Regulators

miRNA mimic

miRNA

1

2

3

4

1) Treat Cells
2) Isolate RNA
3) First Strand cDNA Synthesis
4) qPCR for Gene Expression

- 60 -


Transcriptome cDNA panels
Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the
miRNAs, Pathways or Transcription Factors that regulate the expression of any
gene.

.

Each Transcriptome PCR Array is a panel of Transcriptomes or cDNAs:
90 Experimental cDNA samples
4 Vehicle Control cDNA samples
2 PCR Controls (only for SYBR green reactions)

.

Each experimental cDNA sample is derived from cells treated with a different
treatment.
.

- 61 -


Example Transcriptome PCR Array

cDNA Sample 1
cDNA Sample 2
cDNA Sample 3
cDNA Sample 4
cDNA Sample 5
cDNA Sample 6

- 62 -


Materials needed for miRNA discovery
.

.

.

Transcriptome PCR Array (1 for each GOI or HKG)
The Research Also Needs:
qPCR Thermocycler

Micropipettor
qPCR Master Mix
qPCR Assay (either SYBR® green or Probe-based Assays)
Computer with Microsoft Excel for Data Analysis

- 63 -


SureFIND Protocol

- 64 -


Application Example: miRNA regulation of INPPL1
INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

.

Converts PIP3 to PtdIns(3,4)P2, which negatively regulates PI3K signaling

.

Linked to Carcinogenesis

.

- 65 -


Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

miR-342-3p

miR-184

miR-642

miR-18ab

miR-671-3p

miR-205
miR-27ab
miR-29abc

- 66 -


Cancer miRNA Transcriptome
PCR Array Results for INPPL1

- 67 -


Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

miR-342-3p

miR-184

miR-642

miR-18ab

miR-671-3p

miR-205
miR-27ab
miR-29abc

- 68 -


Compare Results with Research Community

What’s really exciting….
Three new miRNAs that play a role in INPPL1 expression
mir-193b
mir-138
mir-373

- 69 -


Now what?............. Suggestions to Validate your “Hits”
miRNA-based Discovery
Perform a miScript miRNA mimic experiment
Identical Gain-Of-Function experiment
Perform a miScript miRNA inhibitor experiment
Loss-of-Function Experiment
Peform a miScript Target Protector experiment
ssRNA that disrupt a specific Target-miRNA interaction

.

- 70 -


Summary: miRNA Analysis: From Cellular to Molecular

Isolation
miRNeasy

miRNA
Studies


Expression
and PCR Assays


Function
Cignal Reporters

- 71 -


Questions, Comments, Concerns or Suggestions?
*SURVEY?
Answers to your technical and biological questions:
US Applications Support

Global Applications Support
SABio@qiagen.com

888-503-3187
̣

support@sabiosciences.com
Samuel Rulli
Samuel.Rulli@QIAGEN.com
Copy of slides? Type slides into questions box or fill out survey on exit
Want someone to call you? Type contact me into questions box
Want to try something? Request demos in the questions box
miRneasy isolation; -miScript miRNA Arrays; miRNA mimics/inhibitors;
target protectors; Cignal Reporter assays; etc.

- 72 -


Mi rna assays 2012

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