Non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have shown aberrant expression profiles in neurodegenerative disorders. This slideshow reviews the roles of lncRNAs and their mechanisms of action in the regulation of neurodegeneration. Learn more about novel solutions to isolate RNAs from blood and cerebral spinal fluid (CSF). A new qPCR-based lncRNA platform for lncRNA detection and profiling is also presented.

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The Central Roles of Non-coding RNAs in
Neurodegenerative Disorders
Wei Cao, Ph.D.
Wei.Cao@qiagen.com
Noncoding RNAsin Neurodegeneration 1
Welcome!
Contact Technical Support:
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Welcome to our three-part webinar series on neurodegeneration
Noncoding RNAsin Neurodegeneration 2
Neurodegenerative disorders: molecular
mechanismsand circulating biomarker discovery –
a three-part webinar series
 Part 1: Molecular Mechanisms of Neurodegeneration
 Part 2: The Central Roles of Non-coding RNAs in Neurodegenerative
Disorders
 Part 3: Circulating Biomarkers forAlzheimer’s Disease

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Legal disclaimer
Noncoding RNAsin Neurodegeneration 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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The study of non-coding RNA: miRNA and lncRNA4
Agenda
Molecular mechanisms of neurodegeneration1
Introduction to non-coding RNA2
Non-coding RNAin neural function and diseases3
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Molecular mechanisms of neurodegeneration
Noncoding RNAsin Neurodegeneration 5
Neurodegeneration is featured by progressive dysfunction
and death of cells in selected areas in the nervous system.
 Alzheimer’s disease (AD)
 Parkinson’s disease (PD)
 Huntington’s disease (HD)
Common molecular mechanisms
 Abnormal protein assemblies (protein misfolding)
 Late-life cell death in adulthood
 Oxidative stress
 Inflammation – induced neurotoxicity
Molecular basis for neurodegeneration
 Gene mutations and accumulation of abnormal proteins and inclusion
bodies are hallmarks in most neurodegenerative diseases
ncRNAs and ncRNA-regulatory processes are important players in
the pathogenesis of neurodegenerative diseases

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Non-coding RNA – the “dark matter” of the genome
 Over 90% of the human genome is actively transcribed, with only
~2% of the genome being protein-coding
 For a long time, little was known of the genome’s other functions,
being referred to as “dark matter” or “junk”
 Non-coding RNAs: small non-coding RNAand long non-coding RNA
Schw arzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426.
Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17
Non-coding RNAs
The proportion of non protein-coding sequences
increases with complexity in eukaryotes
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Short non-coding RNAs – microRNAs (miRNAs)
 Abundant, the most widely studied ncRNAs and roughly 21 nt in size
 Regulate a variety of cellular processes through post-transcriptional
repression of gene expression
 Expression is frequently deregulated in various diseases, therefore has
potential to serve as biomarkers
Long non-coding RNAs (lncRNAs)
 Novel class of RNAs over 200 nucleotides in size
 Regulate protein-coding gene transcription in more complex ways than
miRNAs
 Changes in lncRNA can be correlated with a variety of human diseases
Schw arzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426.
Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17
Non-coding RNAs – miRNA and lncRNA
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Guil. S. and Esteller, M. (2015) “RNA–RNA interactions in gene regulation: the coding and noncoding players.” Trends in
Biochemical Sciences.
RNA–RNA interactions among different RNA species
The central dogma and regulatory complexity
 One gene gives rise to one RNA to produce oneprotein. RNA has more
versatile functions than its protein synthesis role
 RNAs regulate most cell processes, including epigenetic control, gene
transcription, translation, RNAturnover, chromosomal organizationand
genome defense, cellular developmental and proliferation programs
 RNAs always work through interactions with proteins. However, RNA–
RNA interactions, mediated by ncRNA, add another layer of regulatory
complexity
 ncRNAs regulate gene expression. For example, a single miRNA can affect the
expression of more than 100 transcripts
 Control of splicing through direct base pairing with ncRNAs
 ncRNAs control translation
 lncRNA–miRNA interactions: lncRNAs regulate miRNA biogenesis, and miRNAs
regulate lncRNAs
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miRNA biogenesis and regulatory mechanisms
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MicroRNA (miRNA) biogenesis pathway
Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1
Noncoding RNAsin Neurodegeneration
 Transcribedby RNApolymerase 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 the RNase III
Drosha
 Pre-miRNAs are then exported to the
cytosol by exportin 5
 In the cytosol, the RNAse III 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

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miRNA biogenesis and regulatory mechanisms
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MicroRNA (miRNA) biogenesis pathway
Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1
miRNAs
 Highly expressed in the CNS including
the brain and spinal cord
 Key modulators of both CNS
development and plasticity
 Proteins implicated in
neurodegenerative diseases are
involved in multiple steps of the miRNA
biogenesis pathway
 Represent a novel class of therapeutic
targets for neurodegenerative
disorders
FUS
TDP-43
Noncoding RNAsin Neurodegeneration
Atx2

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miRNAs in neurodegenerative disorders
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miRNAs have been implicated in neurodegenerative disorders
Szafranski, K., et al. “Non-codingRNA in neuralfunction, disease, and aging”, Frontiers in genetics , March 2015
miRNAs  As mediators of brain
development and neuronal
differentiation
 Playing roles in neuronal
longevity and survival
 Regulating
neurodegenerativedisease-
associated pathways
 miRNA dysfunction
downstreamof disease-
linked TDP-43 alterations
could represent an
important pathogenic
mechanism in
neurodegenerativedisease
Noncoding RNAsin Neurodegeneration

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The study of non-coding RNA: miRNA and lncRNA4
Agenda
Molecular mechanisms of neurodegeneration1
Introduction to non-coding RNA2
Non-coding RNAin neural function and diseases3
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 lncRNAs are non-protein-codingtranscripts longer than 200
nucleotides in size
 Most lncRNAs are localized in the nucleus, but some are found in the
cytoplasm
 Many lncRNAs are molecularly indistinguishable frommRNAs
 Although some lncRNAs (e.g., MALAT1) are highly abundant
transcripts, many lncRNAs are less so. Low transcription levels do not
necessarily reflect lack of functionality
 May contain a poly-A tail like mRNA
 lncRNAs are typically less conserved across species and often show
low expression levels and high tissue specificity
Schw arzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426.
Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17
Introduction to long non-coding RNAs (lncRNAs)
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lncRNA classification and subgroup
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Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research
Bulletin 97: 69
lncRNAs can exceed 100,000 nucleotides and cover a wide range of gene positions
lncRNAs can be divided into three general categories:
 Transcribed relative to host protein-coding genes
 Transcribed from gene regulatory regions
 Transcribed from the specific chromosomal regions
Intergenic
Intronic
Exonic
Overlapping
Sense
Antisense
Classified based on their relative position to PCG (protein-codinggenes)
Noncoding RNAsin Neurodegeneration

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The molecularfunctions of lncRNAs
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Martin L, Chang HY. (2012) “Uncovering the roleof genomic "darkmatter" in human disease”, J Clin Invest, 122 1589
Noncoding RNAsin Neurodegeneration

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The study of non-coding RNA: miRNA and lncRNA4
Agenda
Molecular mechanisms of neurodegeneration1
Introduction to non-coding RNA2
Non-coding RNAin neural function and diseases3
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lncRNAs in the central nervous system (CNS)
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The complex functions of lncRNAs coincide with the diversity and elaborate
nature of the central nervous system
lncRNAs in brain development
lncRNAs in neural differentiation
and maintenance
lncRNAs in synaptic plasticity,
cognitive function and memory
lncRNAs in aged brain and
neurodegenerative disorders
Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research
Bulletin 97: 69
Noncoding RNAsin Neurodegeneration

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Dysregulated lncRNAs in neurodegenerative diseases
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lncRNAs Associated disease Biological function
BACE1-AS AD
Increase BACE1 mRNA stability through a post-transcriptional feed-forward
mechanism
NAT-Rad18 AD
Downregulate DNA repair protein Rad18, giving the neuron more sensitivity
to apoptosis
17A AD Impair GABAB signalingpathway by decreasing GABAB R2 transcription
GDNFOS AD Modulate the expression of endogenous GDNF in human brain
Sox2OT AD, PD Regulate co-transcribed Sox2 gene expression to suppress neurogenesis
1810014B01Rik AD, PD Unknown
BC200 AD, PD
Modulate local proteins in postsynaptic dendritic microdomains to maintain
long-term synaptic plasticity
naPINK1 PD
Stabilize svPINK1 resulting in disturbed mitochondrial respiratory chain,
increase sensitivity to apoptosis
HAR1F HD
Aberrant nuclear-cytoplasmic REST / NRSF trafficking causedby mutated
huntingtin resulting in the aberrant expression of HAR1in striatum
HTTAS HD HTTAS v1 specifically reduces endogenous HTT transcript levels
DGCR5 HD DGCR5 is downstream target of REST in HD
NEAT1 HD Essential for the integrity of the nuclear paraspeckle substructure
Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research
Bulletin 97: 69
Noncoding RNAsin Neurodegeneration

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lncRNAs are essential for brain development
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lncRNA Peril−/− mice have reduced viability and die shortly after birth
Sauvageau M, 2013.
Elife. 2:e01749
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874104/
Noncoding RNAsin Neurodegeneration

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lncRNAs in Alzheimer’s disease (AD)
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Faghihi MA, et al (2008) Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of beta-
secretase. Nat Med. 14(7):723
Modarresi F, et al. (2011) “Knockdown of BACE1-AS Nonprotein-Coding Transcript Modulates Beta-Amyloid-Related Hippocampal
Neurogenesis.” Int J Alzheimers Dis. 2011
 AD is the most common neurodegenerativedisorder
 The pathologic process of AD is not well understood. One of the
main reasons is the amyloid plaques caused by increased levels
of Aβ42
 A series of aberrant lncRNAs have been found in AD patients
 BACE1-AS, a lncRNA transcribed from the antisense protein-coding
BACE 1 gene, is highly expressedin AD patients and directly implicated
in the increasedabundance of Aβ42 in AD
 BACE1-AS increases BACE1 mRNA stability and generates additional
Aβ42 through a post-transcriptional feed-forwardmechanism
Noncoding RNAsin Neurodegeneration

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lncRNAs in Parkinson’s disease (PD)
21Noncoding RNAsin Neurodegeneration
 PD is the most frequent motor disorder, and deep-brain-
stimulation (DBS) treatment alleviates symptoms
 RNAseq experiment:
 PD patients’leukocytes pre- and post-DBS treatment were
compared to healthy controls
 Identified 13 lncRNAs (out of 6,000 lncRNAs) with reduced
expression level in PD patients
 Four lncRNAs, RP4-705O1.1, RP11-533O10.2,RP11-
425I13.3 and RP11-79P5.3 were inversely altered in post-
DBS

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lncRNAs alteration in Huntington's disease (HD) brains
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Rory Johnson. “Long non-coding RNAs in Huntington's disease neurodegeneration”, 2012, Neurobiology of Disease. 46:245
Possible mechanism of lncRNAs in HD
 HD is caused by an expansion of a CAG triplet repeat stretch
within the Huntington gene
 REST (RE1 Silencing Transcription Factor) is a target of the
mutated Huntington gene
 Many lncRNAs are direct targets of and regulated by REST
Noncoding RNAsin Neurodegeneration

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The study of non-coding RNA: miRNA and lncRNA4
Agenda
Molecular mechanisms of neurodegeneration1
Introduction to non-coding RNA2
Non-coding RNAin neural function and diseases3
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Current lncRNA quantification approaches
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Sample
isolation
Amplification qPCR
Data analysis
and
interpretation
miRNeasy
exoRNeasy
Serum / Plasma Kit
miScript miRNA
PreAMP PCR Kit
RT² lncRNA
PreAMP PCR Kit
miScript miRNA
PCR Array
RT2
lncRNA
PCR System
Free data
analysis tool
 RNAseq (whole transcriptome sequencing) – discover new RNAs and splicing variants
 Microarrays – use data analysis approaches to identify lncRNAs
 Real-time PCR based approaches – sensitive and quantitative for low-expressing
RNAs and small gene changes
Test and verify your hypothesis with miScript miRNAPCR
Array and RT2 lncRNAqPCR Assays or Custom PCR Arrays
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Real-time PCR quantification of non-coding RNAs
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 The gold standard for gene quantification
 The method of choice to confirm next-generation sequencing
and microarray results
 Simple and easy to carry out
 High sensitivity and specificity
 High throughput compatible, automatable
 Very low template amounts necessary
Noncoding RNAsin Neurodegeneration

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Solutions to miRNA and lncRNA profiling and detection
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How can you advance your non-coding RNA research?
Noncoding RNAsin Neurodegeneration

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miRNA expression — miScript miRNA PCR Arrays
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 miRNome
 Human: miRBase v21, covers 2,402 primer assays
 Mouse: miRBase v21, covers 1,765 primer assays
 Rat: 653 primer assays
 Dog: 277 primer assays
 Rhesus macaque: 469 primer assays
 Cow: 744 primer assays
 Pathway-focused arrays (over 20 arrays)
 miFinder
 Neurological development and disease
 Neuropathic and inflammatory pain
 Apoptosis
 Cell development and differentiation
 Brain cancers
 Serum and plasma miRNAs
 miScript PreAMP Kit
 Optional step for small or precious samples
 Full miRNome profiling from as little as 1 ng RNA
http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-mirna-pcr-arrays
Pre-formatted, single-use PCR arrays with wet lab-verified assays
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lncRNA databases – world wide efforts
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The authoritative lncRNA databases
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 QIAGEN has merged these two databases together for the most up-
to-date qPCR assaydesign
 Currently covers human GENCODE 19, mouse GENCODE M2 and
RefSeqRelease 65
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RT2 lncRNA qPCR system
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 lncRNAdatabases: in-house database at QIAGEN GeneGlobe covers
over 28,000 human and 16,000 mouse lncRNAtargets
 RT2 lncRNAassays: laboratory-verifiedfor optimal qPCR performance
– high specificity, amplification efficiency and sensitivity
 RT2 lncRNAqPCR Arrays: Pathway- or disease-relevant lncRNA
assays
 RT2
lncFinder PCR Array (human and mouse)
 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/
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RT2 lncRNA qPCR Array layout and controls
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Flexible layout and patented controls
Each 96-well plate contains:
 lncRNA-specific assays (84)
 Reference genes (5) (ACTB, B2M, RPL0, RNA7SK, SNORA73A)
 Genomic DNA control (1)
 Reverse transcription controls (3)
 PCR controls (3)
Arrays are also available in 384-well plates and 100-well ring discs for the
Rotor-Gene Q
Free online analysis tool
http://www.qiagen.com/us/landing-pages/lncrna/
Species
Number of qPCR assays designed
(custom designs not included)
Human 42,096
Mouse 27,425
384-well format: 96x4
96-well format
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Custom lncRNA PCR arrays – design your own panels
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Email: BRC.Custom@qiagen.com
Flexibility Cross product
Custom array
(modification)
One to four genes
mRNA and lncRNA
mix ok
Custom array
Flexibility with format
limitation
mRNA and lncRNA
mix ok
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Experiment design: detect and profile lncRNAs
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2. cDNAsynthesis: convert RNAto cDNA
3. Pre-amplification with RT2 PreAMP cDNAkit
1. Isolate total RNA
4.
5.
Samples:
 Control: healthy donor samples
 Patient sample
1. Total RNA isolation
2. cDNA synthesis: convert to total RNA to cDNA using RT2
cDNA
Synthesis Kit
3. Pre-amplification: pre-amplify the target lncRNAs with pre-
amplification RT2
PreAMP Primer Mixes
4. Run PCR: detect lncRNAs with qPCR
 Human RT2 lncRNA PathwayFinder PCR Array
 Master mix: RT2
SYBR®
Green qPCR Mastermix
 qPCR cyclers: any instrument, such as RotorGene-Q or ABI
7900HT
5. Data analysis: GeneGlobe Data Analysis Center
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RT2 lncRNA PCR Arrays
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Compatible with a wide range of qPCR instruments
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We provide service – send samples to us and receive results
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. Whole genome
 Illumina Gene Expression Profiling
 Illumina Genotyping
. Pathway-focused panel
 Mutation profiling
 Methylation
 PCR array
 lncRNA PCR array
 miRNA PCR array
 NGS
. Individual gene / locus
 Mutation detection
 Methylation
 qPCR
 NGS
. Sample preparation – DNA/ RNAextraction and
purification
 Cells, tissue or biofluids
 Fixed tissue
 Small sample
http://www.qiagen.com/products/catalog/services/
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Solutions at QIAGEN
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Non-coding RNAexpression
 miScript miRNA PCR Arrays and Assays
 RT2 lncRNAPCR Arrays and Assays
 Custom PCR Arrays
Protein
 EpiTect ChIP Arrays
 ELISArray kits
Functional studies
 Cignal Reporter Assays
 shRNAand siRNA
Service Solutions
Noncoding RNAsin Neurodegeneration

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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?
Thank you for attending
Noncoding RNAsin Neurodegeneration
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
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