This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.

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Coding and Noncoding RNA Expression Analysis
Coding and NoncodingRNA Expression Analysis 1
Wei Cao, PhD

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Welcome to our four-part webinar series on qPCR
2
qPCR technology overview, applications, data
analysis and service solutions
• Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR)
• Part 2: Advanced Real-Time PCR Array Technology – Coding and Noncoding RNA
Expression Analysis
• Part 3: PCR Array Data Analysis Tutorial
• Part 4: Accelerate Your Discovery With QIAGEN Service Solutions for Biomarker
Research

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Legal disclaimer
Coding & NoncodingRNA Expression Analysis 3
QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
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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Gene expression regulates biology
Coding & NoncodingRNA Expression Analysis 4
All of these pathways require molecular signaling for their activity

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Biological markers define processes
Coding & NoncodingRNA Expression Analysis 5
Pathway-focused solutions for expressionanalysis
Cell cycle Angiogenesis
p53
MDM2
Cyclins
CDKs
VEGF
bFGF
ANGPT
PDGF
IL8
TLR
IFNγ
TNF
Individual
participants
Inflammation

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Biological markers define processes
Coding & NoncodingRNA Expression Analysis 6
Pathway-focused solutions for expressionanalysis
Cell cycle Angiogenesis
p53
MDM2
Cyclins
CDKs
VEGF
bFGF
ANGPT
PDGF
IL8
TLR
IFNγ
TNF
Individual
participants
Inflammation
lncRNAs+lncRNAs+ lncRNAs+

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A complete biological story
Coding & NoncodingRNA Expression Analysis 7
Built on pathway/network analysis
Cell cycle Angiogenesis Inflammation
Pathway

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Differential gene expression analysis
Coding & NoncodingRNA Expression Analysis 8
Question:
How can you assess the expression of different mRNAs in a
sample involved in a process (e.g., angiogenesis, cell cycle, or
inflammation) and compare it across multiple conditions?
RT2 Profiler lncRNAArrays
Answer:

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QIAGEN PCR arrays for all biomedical researchers
Coding & NoncodingRNA Expression Analysis 9
RT2 Profiler PCR Arrays for 13 species powered by over 170 pathways
Cancer and apoptosis Cytokines and inflammation Development and stem cells
Apoptosis Inflammatory cytokines Stem cells
Cell cycle Th17 for inflammation WNT signaling / notch signaling
Human miRNA array Common cytokines / chemokines Terminal differentiation markers
Breast cancer and estrogen receptor Inflammasomes TGFb / BMP signaling
Tumor metastasis NF−κB signaling pathway Endothelial cell biology
Epithelial-to-mesenchymal transition Th1-Th2-Th3 Osteogenesis
Angiogenesis TNF ligands Growth factors
Cancer drug resistance Toll-like receptors ECM and adhesion
Signal Transduction Toxicology and Drug Metabolism Neuroscience
Signal Transduction PathwayFinder Drug metabolism / drug transporters Neuroscience ion channels
NF−κB Signaling Drug phase I enzymes Neurotransmitter receptors
JAK−STAT Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins and receptors
DNA damage signaling Oxidative stress Neurogenesis and neural stem cell
Insulin signaling Stress and toxicity Parkinson’s disease
MAP kinase signaling Other Diseases Custom PCR Arrays (H/M/R/Q/D/F/P/B)
cAMP / calcium Signaling Atherosclerosis 96-well, 384-well plates
p53 Signaling Diabetes 100-well Disc, 96 × 96 chip

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QIAGEN PCR arrays for all biomedical researchers
Coding & NoncodingRNA Expression Analysis 10
The New GeneGlobe − your new research companion! Click “Browse by Research Area”

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Apply your PCR arrays4
Agenda
11
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5

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Characteristics of a good qPCR Assay
Coding & NoncodingRNA Expression Analysis 12
• Amplification efficiency: exponential phase is 100%
efficient
• Sensitivity: able to detect as few as 10–50 copies of
template in one reaction
• Specificity: one assay, one target (no off-target
amplification or primer dimers)
Validation of qPCR assays should be done each
time primers are synthesized to ensure reliable performance
Can be the rate limiting step for using qPCR on 100 genes
and as a routine analysis method for a limited number of samples

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Primer assays
Coding & NoncodingRNA Expression Analysis 13
Laboratory verificationdata:
• Melting / dissociationcurve
o Single peak
• Amplification plot
o PCR efficiency
If you are designing your own primers – stop!
Every primer is experimentallyverified

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Anatomy of an RT2 Profiler PCR Array
Coding & NoncodingRNA Expression Analysis 14
• Rows A–G hold your 84 pathway-specific
genes of interest
• Five housekeeping genes
• Genomic DNA contamination control
(GDC)
• Three reverse transcription controls
(RTCS)
• Three positive PCR controls (PPCs)
• Customizable – Add four genes to a
catalog RT2 Profiler PCR Array or
completely customize based on your
research
Each well contains lyophilized, verified qPCR assays
ACTB, B2M, GAPDH, HPRT1, RPL0

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qPCR primer assays and custom PCR arrays
Coding & NoncodingRNA Expression Analysis 15
Extensive species coverage
Cow (Bos taurus) Chicken (Gallus gallus)
Horse (Equus ferus caballus) Zebrafish (Danio rerio)
Dog (Canis lupus familiaris) Pig (Sus scrofa)
Fruit fly (Drosophila melanogaster) Rabbit (Oryctolagus cuniculus)
Chinese hamster ovary (CHO) cells
(Cricetulus griseus)
Rhesus macaque (Macaca mulatta)
https://www.qiagen.com/us/products/gene and pathways/species-portal/
Wet-bench-verifiedassays for all genes in:
• Human (H / HS)
• Mouse (M / MM)
• Rat (R / RN)
At GeneGlobe, click “Browse by Species”
CustomPCR arrays are expandableto the following species:

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How are genes on PCR arrays selected?
Coding & NoncodingRNA Expression Analysis 16
PCR Array contents are relevant
Biologically relevant gene content
• Not simply biochemical pathways or kinase
cascades
✓ Published association with the biological or disease
pathways gathered from overlapping sources
including:
o Multiple publicly accessible databases
o Text mining relevant literature
Technically relevant gene content
o Use genes that are regulated at the RNA level
o Specific feedback from thought leaders
Genes in signaling pathways

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Relevant pathway content on PCR arrays
Coding & NoncodingRNA Expression Analysis 17

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Functional gene grouping
Coding & NoncodingRNA Expression Analysis 18
Inflammatory cytokines and receptors

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Relevant pathway content on PCR arrays
Coding & NoncodingRNA Expression Analysis 19
Inflammatory cytokines and receptors

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Relevant pathway content on PCR arrays
Coding & NoncodingRNA Expression Analysis 20
Inflammatory cytokines and receptors

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Search by gene, pathway or biology
Coding & NoncodingRNA Expression Analysis 21
https://www.qiagen.com/us/geneglobe/QIAGEN GeneGlobe

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Apply your PCR arrays4
Agenda
22
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5

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Principles of qPCR: an overview
Coding & NoncodingRNA Expression Analysis 23
Real-time PCR – amplify and simultaneously quantify target DNA
• qPCR (reverse transcription real-time PCR)
o Amplify and simultaneously quantify mRNA
• Ct values: threshold cycle
DNA template
(ss or ds)

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Anatomy of an RT2 Profiler PCR Array
Coding & NoncodingRNA Expression Analysis 24
• Rows A–G hold your 84 pathway-specific
genes of interest
• Five housekeeping genes
• Genomic DNA contamination control (GDC)
• Three reverse transcription controls (RTCS)
• Three positive PCR controls (PPCs)
• Customizable – Add four genes to a
catalog RT2 Profiler PCR Array or
completely customize based on your
research
Each well contains lyophilized, verified qPCR assays
ACTB, B2M, GAPDH, HPRT1, RPL0

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How RT2 Profiler PCR Arrays work
Coding & NoncodingRNA Expression Analysis 25
Experimental workflow
Stimulate cells Isolate RNA
qPCR
Convert total RNA to
cDNA
Treat cells, e.g., non-cancer
or cancer cells
Isolate RNA (RNeasy Kits)
RNase-free DNase treatment
Genomic DNA removal step
Reverse transcription step
5 min + 20 min
SYBR Green Mastermix
Real-time PCR detection
2 hr
Control Sample
Data analysis
Upload raw data (Ct) and analyze data
15 min
Data interpretation

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Interpretation of Ct values
Coding & NoncodingRNA Expression Analysis 26
Fold-change calculations for PCR array results/ data
Fold change results per gene = 2-∆∆Ct
Ex: My gene is present 4x more in the treated sample than in control sample.
Now not only one gene, but genes in a pathway (some more, some less)
 Free complete and easy data analysis with web-based software
1. Raw CT value – relative expression level of that gene in the sample
2. Data normalization = ∆Ct per gene = Ctexperiment
– Ctcontrol
3. Upregulation / downregulation = ∆∆Ct per gene = ∆Ctexperiment – ∆Ctcontrol

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RT2 Profiler PCR Array data analysis
Coding & NoncodingRNA Expression Analysis 27
Free complete and easy analysis with Microsoft Excel or web-based software
• Multiple analysis formats support raw Ct analysis, fold change results, and more
Volcano plotScatter plot Clustergram
Two samples
Attend our “PCR Array Data Analysis Tutorial” webinar

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Compatibilityof QIAGEN PCR arrays
Coding & NoncodingRNA Expression Analysis 28
Use QIAGEN PCR arrays with all sample types
Option Sample types Product Cat. no.
1 Cells and easy-to-lyse tissues RNeasy Plus Mini Kit (50) 74134
2
Cells and tissues
with miRNA
miRNeasy Mini Kit (50) 217004
3 Difficult-to-lyse tissues RNeasy Plus Universal Mini Kit (50) 73404
4 Human blood PAXgene Blood RNA Kit (50) 762164
5 Animal blood RNeasy Protect Animal Blood Kit (50) 73224
6 Cells and easy-to-lyse tissues AllPrep DNA/RNA Mini Kit (50) 80204
7 Cells and all tissues AllPrep DNA/RNA/miRNA Universal Mini Kit (50) 80224
8 FFPE samples
RNeasy FFPE Kit (50) 73504
miRNeasy FFPE Kit (50) 217504
9
Exosome-derived RNA from serum
/ plasma
exoRNeasy Serum/Plasma Maxi Kit (50) 77064
Notes:
 Option 3: A phenol-free alternative for fibrous tissues only: RNeasy Fibrous Tissue Mini Kit (50) (cat. no. 74704).
 Accessory product: Rnase-Free DNase Set (50) (cat. no. 79254)

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Compatibilityof QIAGEN PCR arrays
Coding & NoncodingRNA Expression Analysis 29
All qPCR instruments, 96-, 100-, 384- and 96x96-well formats
Manufacturer Instruments*
Applied Biosystems • 96-well blocks: 7000, 7300, 7500, 7700, 7900HT,ViiA 7, QuantStudio
• FAST 96-well blocks: 7500, 7900HT, Step One Plus, ViiA 7, QuantStudio
• FAST 384-well blocks: 7900HT,ViiA 7, QuantStudio
Bio-Rad • iCycler, MyiQ, iQ5, CFX96, CFX384, CFX Connect
• Opticon, Opticon 2, Chromo4
Eppendorf Mastercycler ep realplex 2 / 2S / 4 / 4S
Fluidigm BioMark
QIAGEN Rotor-Gene Q, Rotor-Gene 6000
Roche LightCycler 480, LightCyler 96
Stratagene Mx3000p, Mx3005p, Mx4000p
TaKaRa TP-800 / TP-900 (Dice)
qPCR Results are independent of your PCR instrument. Your results can be replicated!
We provide service! Contact BRC.Service@qiagen.com

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Apply your PCR arrays4
Agenda
30
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5

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Performance of PCR arrays
Coding & NoncodingRNA Expression Analysis 31
Every individual qPCR assayis wet-bench-verified for:
• Sensitivity (25 ng – 5 µg)
o Enhanced to <1 ng with RT2 PreAMP technology
o Compatible with WTAtechnology – down to single cells!
• Specificity
• Reproducibility
• Amplification efficiency
• Wide dynamic range
Reproducibility among different users and different instruments

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Uniformly high amplification efficiency(AE)
Coding & NoncodingRNA Expression Analysis 32
Why does this matter?
Cycle
Gene 1: Your design
(AE=100%)
Gene 1: Your colleague’s design
(AE=85%)
0 1 1
1 2 1.85
2 4 3.42
…
35 3.4x1010 2.2 x109
Δ 15x difference
This differencedoes not reflect biology — it is a technical issue
However, you would have reported it as a biological finding!
Conclusion: Using PCR arrays with uniformly high amplification efficiency, accurate
and reliable ΔCt values are guaranteed

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Replicates: technical and biological
Coding & NoncodingRNA Expression Analysis 33
Technical
• Not necessary – save time and money
• Reproducibility of PCR arrays is very high
• Results demonstrate that what you are seeing is a result of biology, not technique
• RTC and PPC show technical reproducibility on each plate, and comparable results
across plates
Biological
• Needed to verify the results are a result of biology
• At least three replicates per condition for statistical analysis (p values)

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Apply your PCR arrays4
Agenda
34
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5

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Application one: apoptosis resistance
Coding & NoncodingRNA Expression Analysis 35
rhTRAIL
Most cells apoptotic
Few cells resistant to apoptosis
Cancer cells
Translating gene expression into pathways leads to molecular mechanisms
Experimental setup and researchgoals:
• What gene expression changes occur in resistant cells?
• How are these gene changes driving apoptosis resistance?
• Can this explain the mechanism of apoptosis resistance?

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Application one: experimental setup
Coding & NoncodingRNA Expression Analysis 36
• Use QIAGEN sample prep for RNA purification
(recommend miRNeasy kits for mRNA/lncRNA
and future miRNA)
• Use RT2 First Strand Kit for cDNA conversion
o Integrated DNase step
o Proprietary spike in RNA
o Priming with both oligo-dTs and
random hexamers
• Use RT2 SYBR Green master mix for qPCR
with RT2 Profiler PCR Array (RT2 Profiler
Apoptosis PCR Array (384-well))
• Go to QIAGEN’s Data Analysis Center for
analyzing lncRNA expression data
Separate
beads
Experimental setup MDA-MB-231cells

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Application one: results
Coding & NoncodingRNA Expression Analysis 37
Apoptosis
• Caspases and regulators
• Anti-apoptosis
• Pro-apoptosis
• TNF and TNFR domains
• BCL2 and BAG domains
• BIR domain
• CARD domain
• DEATH domain
• TRAF domain
One sample per plate, 370 genes

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Application one: results
Coding & NoncodingRNA Expression Analysis 38
High rhTRAIL
dose
Low rhTRAIL
dose
MDA-MB-231 cells
rhTRAIL resistant
MDA-MB-231 cells:
• Highly sensitive to TRAIL induced apoptosis at optimal TRAIL concentration
• Low doses of TRAIL result in in TRAIL-induced apoptosis-resistant cells
Results:
qPCR reveals up regulation of c-FLIP, Stat5a and Stat5b, Bcl-xL, and cyclin D1
• c-FLIP antagonizes caspase-8 activation
• Stat5b is responsible for Bcl-xL and cyclin D1 transcription
• Bcl-xL is an anti-apoptosis protein
Apoptosis

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Application two: tumor analysis
Coding & NoncodingRNA Expression Analysis 39
Many questions are raised by this experiment:
• What if you knocked down all the upregulated
genes and induced all the downregulated genes?
o Would the cells revert to normal?
• Why are TIMP3 and MMP3 upregulated
together?
o And why at different rates?
• What correlation might there be between MMP3
and MMP9?
The extracellular matrix and adhesion molecules:
• RT2 Profiler PCR Array revealed up- and
downregulated genes in breast cancer
Spend your time analyzing and
thinking about your data!

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Introduction to long non-coding RNAs (lncRNAs)
Coding & NoncodingRNA Expression Analysis 40
Long non-coding RNAs(lncRNAs)
• lncRNAs are a novel class of RNAs >200 nucleotides in size
• Many lncRNAs are molecularly indistinguishable frommRNAs
• lncRNAs regulate protein-codinggene transcription in complex ways
• Changes in lncRNAcan be correlated with a variety of human
diseases
• Most lncRNAs are localized in the nucleus with some found in the
cytoplasm
• Abundance: Some lncRNAs (e.g., MALAT1) are highly abundant
transcripts, but many lncRNAs do show a low count. Low transcription
levels do not necessarily reflect a lack of functionality
• May have a poly-Atail like mRNA
• lncRNAs tend to be less conserved acrossspecies, often showinglow
expression levels and high tissue specificity

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Molecular functions of lncRNAs
Coding & NoncodingRNA Expression Analysis 41

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Expanding the RNA Universe! RT2 lncRNA qPCR system
Coding & NoncodingRNA Expression Analysis 42
• lncRNAdatabases: the in-house database at QIAGEN GeneGlobe
provides over 28,000 human and 16,000 mouse lncRNAtargets
• RT2 lncRNAassays: assays laboratory-verified for optimal qPCR
performance — high specificity, amplification efficiencyand sensitivity.
• RT2 lncRNAqPCR Arrays: pathway- or disease-relevant lncRNA
assays
• Custom option: flexible custom design from the lncRNAdatabase and
qPCR database to profile mRNA and lncRNAsimultanously
• lncRNAisolation: miRNeasy kits or exoRNeasy kits
• Data analysis: free online data anlysis tool
http://www.qiagen.com/us/landing-pages/lncrna/
lncRNA research is now possible for any lab with RNA or cDNA

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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 43
• Prostate cancer is the most common cancer in American men
• Estimated new cases in 2014: 233,000
• Estimated deaths in 2014: 29,480
• Stages
o Prostate cancer stage I: Found in the prostate only. Stage I prostate
cancer is microscopic; it can’t be felt on a digital rectal exam (DRE),
and it isn’t seen on imaging of the prostate
o Prostate cancer stage II: the tumor has grown inside the prostate but
hasn’t extended beyond it
o Prostate cancer stage III: Prostate cancer has spread outside the
prostate, but only barely. Prostate cancer in stage III may involve
nearby tissues, like the seminal vesicles
o Prostate cancer stage IV: The cancer has spread (metastasized)
outside the prostate to other tissues. Stage IV prostate cancer
commonly spreads to lymph nodes, the bones, liver or lungs
http://www.qiagen.com/us/landing-pages/lncrna/

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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 44
• Key questions:
o Can lncRNA be detected with RT2 lncRNA PCR
Arrays?
o Will literature-verifiedlncRNA expressionchange be
confirmed with RT2 lncRNAArray?
o Will there be any new links of lncRNA to prostate
cancer?
o Will there be any stage-relatedlncRNA changes
that have potential as biomarker?
http://www.qiagen.com/us/landing-pages/lncrna/

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Application three: materials and methods
Coding & NoncodingRNA Expression Analysis 45
• Samples (total RNA from fresh frozen tissue sample, reverse
transcription with RT2 First strand kit):
o Control Group: Normal control samples
o Group 1: Prostate cancer Stage II
o Group 2: Advanced Prostate cancer – Stage III-IV
• PCR Array-RT2 lncRNA PCR Array-Human Cancer
PathwayFinder (84 cancer related lncRNA assay with
controls in 96-well plate).
• Master mix: RT2 SYBR Green ROX™ qPCR Mastermix
• qPCR cyclers: ABI 7900HT
• Data analysis: GeneGlobe Data Analysis Center
http://www.qiagen.com/us/landing-pages/lncrna/

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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 46
Cancers:
Bladder cancer: BLACAT1, H19, MALAT1, MEG3, SNHG16, TERC, TUG1, UCA1
Breast cancer: BCAR4, GAS5, H19, HIF1A-AS2, HOTAIR, JADRR, LSINCT5,
MIR31HG, MRPL23-AS1, TRERNA1, UCA1, XIST, ZFAS1
Cervical cancer: MEG3
Colorectal cancer: CCAT1, CCAT2, CRNDE, H19, HOTAIR, MALAT1, PCAT1, PVT1,
SNHG16, TUSC7
Endometrial cancer: H19, HOTAIR
Esophageal squamous cellcarcinoma: CBR3-AS1, HOTAIR
Gallbladder cancer: MALAT1
Gastric cancer: CCAT1, CDKN2B-AS1, GACAT1, H19, HOTAIR, HULC, LINC00152,
LINC00261, LINC00312, MEG3, PVT1, SUMO1P3, TERC
Glioma: H19, HOTAIR, MEG3
Kidney cancer: AFAP1-AS1, DGCR5, HIF1A-AS2, WT1-AS
Laryngeal squamous cell carcinoma: HOTAIR, MALAT1
Leukemia: DLEU2, GAS5, H19, HOTAIR, HOXA-AS2, MALAT1, MEG3, TERC, TUG1,
XIST
Liver cancer: H19, HEIH, HOTAIR, HOTTIP, HULC, KCNQ1OT1, MALAT1, MEG3,
MIR7-3HG, PANDAR
Non-small cell lung cancer: BANCR, CCAT2, GAS6-AS1, HOTAIR, MALAT1, MEG3
Other lung cancers: ACTA2-AS1, LUCAT1
Melanoma: BANCR, HOTAIR, PTENP1
Multiple myeloma: MEG3, PVT1
Nasopharyngeal carcinoma: HOTAIR, LINC00312
Neuroblastoma: HAND2-AS1, SNHG16
Oesophageal squamous Cell Carcinoma: LINC01234
Osteosarcoma: TUG1
Ovarian cancer: H19, HOTAIR, LSINCT5, WT1-AS, XIST
Pancreatic cancer: GAS5, HOTAIR, PVT1
Pituitary cancer: MEG3
Prostate cancer: CBR3-AS1, GAS5, HOTAIR, LINC00963, MALAT1, PCA3, PCAT1,
PCGEM1, PRNCR1, PVT1, TERC, XIST
Rhabdomyosarcoma: RMST
Sporadic pediatric adrenocortical tumors: KCNQ1OT1
Testicular cancer: XIST
Thyroid cancer: NAMA, PTCSC3
Oncogenes: ACTA2-AS1, AFAP1-AS1, BANCR,
BCAR4, BLACAT1, CBR3-AS1, CCAT1, CCAT2,
CRNDE, H19, HAND2-AS1, HEIH, HIF1A-AS2,
HNF1A-AS1, HOTAIR, HOTTIP, HOXA-AS2, HULC,
JADRR, KCNQ1OT1, LINC00152, LINC00963,
LSINCT5, LUCAT1, MALAT1, MIR155HG, MIR17HG,
PCA3, PCAT1, PCGEM1, POU5F1P5, PRNCR1,
PVT1, SNHG16, SPRY4-IT1, SUMO1P3, TERC,
TRERNA1, TUG1, UCA1, XIST
Tumor suppressor genes: ADAMTS9-AS2, CAHM,
DLEU2, DLX6-AS1, GACAT1, GAS5, GAS6-AS1,
GNAS-AS1, LINC00261, LINC00312, MEG3,
MIR31HG, MIR7-3HG, NAMA, NEAT1, PTCSC1,
PTCSC3, PTENP1, TERC, TUSC7, WT1-AS, ZFAS1
Other cancer-related lncRNAs: AIRN, EMX2OS,
FTX, HIF1A-AS1, HOTAIRM1, HOXA11-AS, IPW,
KRASP1, LINC00538, LINC00887, LINC01233,
NBR2, NRON, RMRP, RPS6KA2-AS1, TSIX

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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 47
RT2 lncRNACancer PathwayFinderPCR Array provides an easy approach for lncRNA profiling. In
prostate cancer samples the well known biomarker PCA3 was easily identified. At the same time,
novel tumor suppressor ADAMTS9-AS2 was also linked to prostate cancer progression.
Group one (prostate cancer (PCa) stage II) vs. control group (healthy controls)
Prostate cancer samples
showed significant
lncRNAexpression
changes compared with
normal controls
ADAMTS9-AS2
PCA3
Down Up
5-fold up-regulation
P value <0.01

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Application three: significant changes that correlate with PCa stage III/IV
Coding & NoncodingRNA Expression Analysis 48
By quickly screening more samples, the novel tumor suppressorlncRNAADAMTS9-AS2 was shown
to be further downregulated in stage III/IV prostate cancer samples
Is ADAMTS9-AS2 a new biomarker?More samples are needed
ADAMTS9-AS2 was shown to be further downregulated in stage III/IV
prostate cancer samples

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Apply your PCR arrays4
Agenda
49
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5

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What comes after RT2 Profiler PCR Arrays?
Coding & NoncodingRNA Expression Analysis 50
• Verify results with more samples and a focusedset of genes
o RT2 qPCR Primer Assays or RT2 qPCR lncRNAPrimer Assays
o RT2 Profiler Custom PCR Arrays
• miRNA studies
o miScript miRNA PCR Arrays and qPCR Assays
• Assess biological impact
o Cignal Reporter Assays and Cignal Finder Reporter Arrays
• Quantify secretedproteins in blood, plasma and sera
o ELISArrays
• Knockdown analysis
o SureSilencing shRNAPlasmids and FlexiTube and FlexiPlate siRNA

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RT2 Profiler PCR Array system summary
Coding & NoncodingRNA Expression Analysis 51
• Complete experimental systems
o RNA isolation kits, PCR arrays, master-mix, first strand kit and free data analysis software
o Systematic controls to monitor sample quality, reverse transcription and PCR efficiency
o RNA to data analysis in only two hours
o Compatible with RNA-seq and microarray experiments but more focused
o Generate statistics – p values help justify significance of small gene expression changes
• Breadth of pathway content
o Profile the genes and pathways that you really care about
o Over 150 pathways for human, mouse, rat, Drosophila, rhesus macaque, dog, pig and more
o Customizable by gene, set or pathway
• Laboratory-verifiedqPCR performance on every lot of qPCR assays
o Amplification efficiency + specificity + high sensitivity = Reliability
• Applications by pathway, disease or expand upon single gene experiments

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Thank you for attending!
Coding & NoncodingRNA Expression Analysis 52
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCsupport@QIAGEN.com
Wei Cao, Ph.D.
Wei.Cao@QIAGEN.com
Questions?
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.