Mi rna array 2013

miScript miRNA PCR Arrays

Simultaneous Detection of Genome-Wide or
Pathway-Focused miRNA

Samuel Rulli, Ph.D.
Samuel.Rulli@QIAGEN.com
support@sabiosciences.com (US)
SAB.EU@qiagen.com (international)
Sample & Assay Technologies

Topics to be Covered

Introduction to miRNA
miScript miRNA PCR Array Overview
miRNA isolation
miScript miRNA PCR Arrays and Assays
Pre-amplification of miRNA
Data Analysis

miScript miRNA PCR Array Applications
Cancer
Development & Differentiation
Genome-Wide Discovery

-2-


What Is MicroRNA?
Endogenously expressed small functional RNAs (19-25 nt)

.

Regulate mRNA expression post-transcriptionally
mRNA degradation, but also translation inhibition

.

Only partial sequence complementarity required
One miRNA can potentially regulate hundreds of mRNAs
One mRNA can be regulated by multiple miRNAs

.

Another layer of complexity for regulating gene expression

-3-


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 IIIlike 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
Krol, J et.al., (2010) Nature Rev Genetics, 11, 597; Winter, J. et.al., (2009) Nature Cell Biology, 11, 228
-4-


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

-5-


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

-6-


miRNAs are continually being discovered
miRNA database: www.mirbase.org
New miRNAs continually discovered
(release 19.0, August 2012)

.

.

.

Human: 1066 (V16) 2032 (v19)
Mouse: 940 (v16) 1281 (v19)
Rat: 653 (v16) 721 (v19)
Nomenclature
Pri-miRNA (>100nt)
Pre-miRNA (~70nt)
Mature miRNA (~20nt)

.

-7-


miRNA Research is an exponentially growing field

HighWire + PubMed Publications

miRBase Entries
25000

mature miRNA sequences

miRNA Publications

12000
10000
8000
6000
4000
2000
0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

* Projected

*

Year

20000

15000
10000

5000
0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18

miRBase Version

Virtually every publication includes quantification experiments.
Changes in miRNA correlated with gene expression changes in development,
differentiation, signal transduction, infection, aging, and disease.

-8-


Why Analyze miRNA Expression Patterns?
Potential for genome-wide coverage, even though >1000 miRNAs
identified

.

miRNA expression profiles correlate with:
Protein & Gene Expression Levels, Biological Phenotypes

.

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

-9-


Unique miRNA signatures are found in human cancer
(first studies around 2005)
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.

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.

The race is on to develop miRNA biomarkers and therapeutics!

- 10 -


Circa 2011
A growing picture of miRNA dysregulation in cancer

Deletions of miRNA-regulated genes have been detected in:
>65% of chronic lymphocytic leukemia cases,
50% of mantle-cell lymphomas,
16% to 40% of multiple myelomas,
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

- 11 -


miRNA expression profiling
Key considerations

Scientific question
Well-defined and testable

Experimental sample set
Statistically meaningful number of replicates (biological replicates)
A minimum of 3 replicates recommended
Replicates enhance the statistical power

Inclusion of proper normalization controls & spike in controls in the analysis

Experimental testing platform
Simple, straightforward workflow
High sensitivity & specificity
A variety of PCR arrays to meet your experimental needs
Easy and simple data analysis tools
Customizable solutions & single assays
Availability of companion research tools

- 12 -


How to Monitor miRNA Expression Levels
Northern Blot
Very low throughput
Very time consuming and complex

.

Microarrays
Profile More Sequences
Poor Sensitivity & Dynamic Range, Complicated Protocol

.

Next-gen sequencing
Quantifiable?
Discover novel sequences, but need validation
Cost for routine analysis of 12+ samples (complex procedures, $$ for instrument)

.

Real-Time RT-PCR
Profiles Fewer Sequences (Up to 384)
Simpler Protocol
Better Sensitivity, Specificity & Reproducibility

.

- 13 -


Topics to be Covered

Introduction to miRNA
miScript miRNA PCR Array Overview
miRNA isolation
miScript miRNA PCR Arrays and Assays
Pre-amplification of miRNA
Data Analysis

miScript miRNA PCR Array Applications
Cancer
Development & Differentiation
Genome-Wide Discovery

- 14 -


miScript miRNA PCR Array System
complete system from isolation to published data
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 and others: Search miRNA Primers:
http://www.qiagen.com/Products/miScriptPrimerAssays.aspx

.

miScript miRNA PCR Arrays: Pathways and Genomes
Optimized miScript SYBR® Green Master Mix

.

miScript miRNA Data Analysis Excel Template & Web Portal

.

- 15 -



.

One-step reaction

.


.


.


.

- 16 -


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

miScript miRNA PCR Assays and
Arrays profile mature miRNA from
TOTAL RNA
.

No need to separate miRNA
Use co-purified Total RNA
Best practice for mRNA and
miRNA detection
Able to use mRNA now and
archive miRNA as cDNA
(increased stability and biobanking)

- 18 -


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

- 19 -


miRNeasy Serum/Plasma Kit Purification of high quality total
RNA from serum or plasma samples

- 20 -



.

One-step reaction

.


.


.


.

- 21 -


miRNA RT-PCR Technical Difficulties

Short sequence (21-23nt)

.

Background from contaminating small RNAs (tRNA, rRNA, snRNA)

.

Highly homologous
Many miRNAs have a single nucleotide difference

.

- 22 -


miScript PCR System
Reverse Transcription using HiSpec Buffer

- 23 -


Performance: Universal RT Advantages
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
cDNA can be pre-amplified allowing analysis from few cells or
amplification of limiting samples for miRNome analysis

.

Same specificity as stem loop primers with additional flexibility

- 24 -



.

One-step reaction

.


.


.


.

- 25 -


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
- 26 -


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

384-well

384-well (4 x 96)

Rotor-Disc 100

- 27 -


miScript PCR Controls
Stable expression and excellent amplification efficiencies

Tissue Expression

35

Mean CT

30

SNORD61
SNORD68

25

SNORD72

20

SNORD95

15

SNORD96A
RNU6-2

10
5
Brain

Kidney

Liver

Lung

Skeletal
Muscle

Testis

Thymus

Amplification Efficiencies
Primer

Hsa

Mmu

Rno

Cfa

Mml

SNORD61

103

99

95

99

101

SNORD68

101

100

93

97

99

SNORD72

94

100

93

91

97

SNORD95

102

103

98

100

106

SNORD96A

102

95

95

92

104

RNU6-2

98

98

95

99

103

Highly conserved across multiple species
Human, Mouse, Rat, Dog, Rhesus macaque

Relatively stable expression in many tissues
Amplification efficiencies of these assays are 100%
Consistent performance in both HiSpec and HiFlex Buffers
Ideal normalizers for ∆∆CT method of relative quantification

- 28 -


Redefining miRNA expression profiling

Benefit of miScript miRNA PCR Arrays and Assays
100% validated assays
Each assay is bench validated
Each array is quality controlled
Leading miRNome coverage
miRNome coverage is continually updated
Validated designs on miRBase V16/V18
Assay nomenclature compatible with miRBase V19
New content added monthly for pathway focused content
Fully customizable
- 29 -


Reliable and routine miRNA expression profiling

Pathway-Focused Arrays

Complete miRNome Arrays

Human, Mouse, Rat:
Human
Mouse
Rat
Dog
Rhesus macaque
Species

miFinder (Dog and Rhesus macaque)
Cancer PathwayFinder
Brain Cancer
Breast Cancer
Ovarian Cancer
Cell Differentiation & Development
Immunopathology
Inflammatory Response &
Autoimmunity
Neurological Development & Disease
T-Cell & B-Cell Activation
Prostate Cancer
Serum & Plasma

Assays
(miRBase V16)

Human

1066

Mouse

940

Rat

653

Dog

277

Rhesus macaque

469 (V18)

- 30 -


Redefining miRNA expression profiling

New! High Content


miFinder 384HC

Human, Mouse, Rat:

Profiles the expression of the 372 most
abundantly expressed and best
characterized miRNAs in miRBase

miFinder (Dog and Rhesus macaque)
Cancer PathwayFinder
Brain Cancer
Breast Cancer
Ovarian Cancer
Cell Differentiation & Development
Immunopathology
Inflammatory Response &
Autoimmunity
Neurological Development & Disease
T-Cell & B-Cell Activation – New!
Prostate Cancer – New!
Serum & Plasma

Serum & Plasma 384HC
Profiles the expression of 372 miRNAs
detectable in serum and plasma using
the miScript PCR System
Cancer Finder 384HC
Profiles 372 miRNAs associated with
various cancers and cancer stages

- 31 -



.

One-step reaction

.


.


.


.

- 32 -


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

.

- 33 -


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

- 34 -


Performance: Reproducibility

The miscript miRNA PCR Assays are highly reproducible,
ensuring run-to-run, plate-to-plate and sample-to-sample
reliability.
- 35 -


Replicates: Technical & Biological
Technical
Reproducibility of the PCR Arrays is very high
Results demonstrate that what you are seeing is a result of biology, not
technique.
RTC & PPC show technical reproducibility on each plate, and comparable
across plates.

Biological
Needed to verify the results are a result of biology
Need multiple samples
At least 3 replicates per sample for statistical analysis
p values
95% Confidence Intervals

- 36 -


Pre-amplification for miRNA detection in limiting samples

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

- 37 -


miScript PreAMP PCR Kit – decrease input up to 1000X
Straightforward, reproducible and robust amplification

Mean CT: Operator 2

30

25

20
y = 0.9973x + 0.2058
2

R = 0.9913
15
15

20

25

30

Mean CT: Operator 1

Universal reference RNA
Two operators
Experienced operator
First time operator
miScript real-time PCR
miFinder miScript miRNA PCR Array

- 38 -


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 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!
- 39 -


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

32

: Colorectal Cancer Serum

1.E+03

28

1.E+02

1.E+01

24

1.E+00

-∆CT

20
y = 0.9728x + 0.7671

2

CT Mean: 3 RNA Isolations from Serum

Reproducibility

2

R = 0.9757

1.E-01

28

32

CT: Individual RNA Isolation from Serum

2

-∆CT

1.E+03

24

1.E+02

20

1.E+01

16

1.E+00

1.E-01

16

: Normal Serum

Workflow
5 µl serum
14 µl total RNA 5 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
- 40 -



.

One-step reaction

.


.


.


.

- 41 -


Data analysis
miScript miRNA PCR Array Data Analysis Tools

- 42 -


Data analysis using free software
miScript miRNA PCR Array Data Analysis Tools

Web-Based Software

.

No installation needed
Tailored for each array

Raw CT Values to Fold-Change Results

.

Using ∆∆CT Method

Other methods available
Multiple Analysis Formats

.

Scatter Plot
Volcano Plot
Multi-Group Plot
Clustergram
.

Downloadable Excel Templates also available

- 43 -


Application Data: Cancer Biomarkers

Human Cancer miscript miRNA PCR Array:
Many Cancer-Specific miRNA Are Up-regulated in a
Colon Tumor.

- 44 -


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 miRNA Potentially
Regulating Tissue-Specific Gene Expression.

- 45 -


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

miscript Human Genome miRNA PCR Array: Identifies Known
and Novel miRNA Targets of the p53 Signaling Pathway

- 46 -


Application Data: FFPE

Expression rank order of 88
cancer related miRNAs in
RNA extracted from one
20µm section of a 2-year
old normal human colon
FFPE block using the
miscript FFPE RNA
Extraction Kit.

- 47 -


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

- 48 -


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 cutoff for significance

- 49 -


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
- 50 -


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!

- 51 -


SUMMARY

miRNA Function

.

• Regulates gene expression post-transcriptionally

miRNA Performance

.

• RT-PCR vs. Microarrays
• Universal vs. Sequence-Specific RT
• Specificity, Sensitivity & Reproducibility

miRNA Applications

.

• Screen cancer- or development-focused miRNA panels
• Profile miRNAs by genome and follow-up with custom miRNA PCR
array panels.

- 52 -


SciGlobe: Search a for your papers using a bigger database than Pubmed and
more relevant than Google Scholar

SciGlobe Advantage
•Free
•Weekly emails on
new papers
•Download and store
papers
•Export to citation
databases
•Larger database than
pubmed
•More relevant
searches than Google
scholar

Try it Now: http://www.sabiosciences.com/sciglobe.php

- 53 -


SciGlobe: Search a for your papers using a bigger database than Pubmed and
more relevant than Google Scholar

SciGlobe Advantage
•Free
•Weekly emails on
new papers
•Download and store
papers
•Export to citation
databases
•Larger database than
pubmed
•More relevant
searches than Google
scholar

Try it Now: http://www.sabiosciences.com/sciglobe.php

- 54 -


Manipulating miRNA function

- 55 -


Mi rna array 2013

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (20)

Similar to Mi rna array 2013

Similar to Mi rna array 2013 (20)

More from Elsa von Licy

More from Elsa von Licy (20)

Mi rna array 2013