total rna discovery for rna biomarker development webinar
TRANSCRIPT
Sample to Insight
Eric Lader, Ph.D.
[email protected] Director, Product Development
Total RNA DiscoveryFor RNA Biomarker Development
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Supporting Sample to Insight
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The products described in this webinar are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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Characteristicpresence of antibodiesabnormal bp, blood cells, electrolytes distinct histological indicatorsabnormal liver or kidney function markerspresence of muscle injury protein markersgene status or gene expression status
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What is a biomarker?
MethodologyElisablood counts, pressuremicroscopybiofluid assaybiofluid assayqPCR, NGS, Array, etc.
A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
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Nucleic Acid biomarkers
Gateway to Personalized Medicine• Stable ‘universal’ analyte• Universal detection technologies• Single molecule sensitivity and specificity
What is theprognosis
What is the right treatment
Monitoring effectiveness
Recurrenceor progression
What kind of cancer is it
Predictive BRCA germline mutationDiagnostic BCR-ABL somatic translocationPrognostic HER2 somatic amplificationTheranostic CYP26 SNP germline variant
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Noninvasive biomarkerIn order to use a biomarker for routine diagnostics, the sample material must be as easy to obtain as possible.
urine or saliva sample
a drop of blood
blood sample
CSF
surgical biopsy
Evaluation
MinimallyInvasive
invasive
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Pubmed: gene expression as ‘biomarker’
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>22,000 peer-reviewed publications on RNA AND biomarker…
“RNA Biomarker”
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RNA in body fluids
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Exosomes and Microvesicles
RNA
Ago
Protein-RNA complexes
High levels of nucleases present in plasma Freely circulating RNA should be rapidly degraded Surprisingly, RNA can be detected in serum and plasma
CTC (in cellular compartment)
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Exosomes harbor unique subsets of RNA and proteins
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Exosomes are small vesicles excreted by all cells, found in all biofluids
Exosomes contain stable nucleic acids (mRNA, microRNA, lncRNA), DNA, and protein, protected from degradation by a lipid bilayer
Contents are specifically packaged, mechanism of local and distant cellular communication
Exosomes can function as remote intercellular mediators
Cancer cells shed large numbers of exosomes which may be critical in disease progression
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Total RNA isolation technologies
miRNeasy family (total RNA from body fluids or cells)
QIAamp Circulating Nucleic Acid Kit(larger input volumes of body fluid if required)
exoRNeasy Serum/Plasma Kit(exosome purification)
Cells CSF Urine
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miRNeasy Serum/Plasma Kit
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RNA isolation from cells, serum, plasma, and other biofluids
Includes synthetic RNA control assay for normalization
1 µl to 400 µl input volume
Optimized elution volume (14 µl)
High-purity RNA suitable for all downstream applications
Easy, robust procedures
Automatable protocol QIAzol Bind Wash Elute
Manual or automated on QIACube
Plasma, Serum, Cells
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exoRNeasy Serum/Plasma Maxi Kit
Microvesicle isolation 20 minutes
RNA isolation 35 minutes
Separate Serum/Plasma
Isolate Exosomes
Isolate RNA
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What RNA species are being developed as biomarkers?
RNA markers 2000: mRNA
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RNA markers 2000: mRNA
RNA markers 2015: mRNA, miRNA, lncRNA
What RNA species are being developed as biomarkers?
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Human genome: 90% transcribed, BUT only 2% translated!
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The “Total RNA Story” is one of complex interaction of coding and non-coding RNAs with each other, with
proteins, and with the genome.
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Non-coding RNA: lncRNA
Long non-coding RNAs (long ncRNAs, lncRNA) are non-protein coding transcripts longer than 200 nucleotides.
There are >20,000 lncRNAs in humans. They are inter-genic, intra-genic, and antisense to traditional mRNA loci.
Do not contain an open reading frame – (or at least aren't translated using standard codons).
May contain a Poly-A or may be absent
lncRNA expression is generally lower than mRNA. Sensitive methods such as qPCR are needed for detection & quantification.
The important central roles of lncRNA in biology and pathology are extremely diverse and currently being elucidated.
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lncRNAs: Broad functionality leads to diverse biological roles
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1. Negatively affect expression2. Positively affect expression3. Hybridize to pre-mRNA resulting in an
alternatively spliced transcript4. Hybridization of sense and antisense transcripts
allow Dicer to generate endogenous siRNAs5. miRNA ‘sponges’
6. The complex of lncRNA and specific protein partners can modulate the activity of the protein
7. Involved in structural and organizational roles in the cell
8. Alters protein localization in the cell9. Affects epigenetic process
Imprinting Cancer and cancer
progression Development Signaling
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Non-coding RNA: miRNAs, Master regulators of gene expression
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miRNAs are ~21 nucleotide, small non-coding RNAs that are conserved in all eukaryotes and expressed in all mammalian tissues.
Changes in miRNA expression can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging, and disease.
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.
Mature miRNAs are incorporated in RISC.
miRNA guide RISC to inhibit translation of, destabilize, or specifically cleave mRNA targets.
~1000-3000 in humans
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Tools for qRTPCR-Based gene expression profiling
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RT2 Profiler and miScript PCR Arrays
Innovative content, extensive species coverage, and formats for every real-time instrument
Biologically relevant gene content Not simply biochemical pathways or kinase cascades Published association with the biological or disease
pathways gathered from overlapping sources, including: Multiple publicly accessible databases Text mining relevant literature
Technically relevant gene content (mRNA) Use genes that are regulated at the RNA level Specific feedback from thought leaders
What species? Human, mouse, rat Cow, horse, dog, chicken, fruit fly, CHO, zebrafish, pig,
rabbit, rhesus macaque What formats?
96-well, 384-well, Fluidigm® BioMarkTM
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Pioneering content to accelerate your hypothesis testing
Pathways Apoptosis Biomarkers Cell Cycle Cytokine & Inflammation ECM & Adhesion Neuroscience Signal Transduction Stem Cell & Development Toxicology & Drug ADME
Diseases Specific Cancers Cardiovascular Diseases CNS Disorders Immune Disorders Infectious Diseases Metabolic Diseases
Targeted miRNomes Targeted miRNomes miFinder 384HC: Best expressed,
best characterized miRNAs Serum & Plasma 384HC: Best expressed
miRNAs in serum/plasma Cancer PathwayFinder 384HC Liver miFinder 384HC
Focused Arrays miFinder Cancer specific arrays Apoptosis Cell Differentiation & Development Diabetes Inflammatory Response & Autoimmunity T-Cell & B-Cell Activation Tumor Suppressor miRNAs Serum & Plasma
mRNA Arrays miRNA Arrays
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RT2 mRNA and lncRNA System, miScript System
Quantify mRNA, lncRNA, miRNA from ANY sample on ANY real-time instrument
1. Isolate total RNA2. Perform Reverse-transcription3. (Optional): Perform PreAMP4. Prepare PCR pre-mix5. Load PCR arrays or plates6. Perform real-time PCR7. Analyze data
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Profiling example: miRNAs present in biofluids
serum 20 ul urine 20ul & preamp csf 20ul & preamp
In each array, expression values normalized to mean of expressed sequences
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RNA expression distinguishes thyroid cancer from benign nodules
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Thyroid nodules and thyroid cancer
Thyroid nodules are very common
525,000 U.S. patients each year undergo fine needle aspiration (FNA) biopsies to rule out cancer in thyroid nodules
Some commercial malignancy classifier assays are available but their performance requires further evaluation
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Profiling workflow
Samples: Frozen thyroid tissue
Benign: Nodular hyperplasia (NH) and follicular adenoma (FA)
Cancer: Papillary thyroid cancer (PTC) and follicular variant of papillary thyroid carcinoma (FVPTC)
Sample prep: miRNeasy Mini Kit
Real-time PCR Assays:
mRNA and lncRNA: RT2 Expression System
mRNA: Thyroid cancer-related mRNAs
lncRNA: Cancer PathwayFinder
miRNA: miScript PCR System
miRNA: miFinder 384HC
Data analysis: GeneGlobe Data Analysis Center
Further interpretation: Ingenuity Pathway Analysis
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mRNA profiling of thyroid nodules and thyroid cancer
68 mRNAs up-regulated
CHI3L1 (Jarzab et al., Cancer Res, 2005)
46 mRNAs down-regulated
cKIT (Marini et al., Journal of Thyroid Research, 2011)
Thyroid cancer-focused array
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miRNA profiling of thyroid nodules and thyroid cancer
17 miRNAs up-regulated
hsa-miR-146b-5p & hsa-miR-221-3p (Chen et al., Mod Pathol, 2008)
hsa-miR-31-5p and hsa-miR-31-3p ~100X up-regulated
4 miRNAs down-regulated
hsa-miR-211-3p (down-regulated in breast cancer)
miFinder 384HC (core miRNome)
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lncRNA profiling of thyroid nodules and thyroid cancer
PTCSC1, papillary thyroid carcinoma susceptibility candidate 1 A susceptibility locus for papillary thyroid
carcinoma on chromosome 8q24
SNHG16 (ncRAN) disregulated in colorectal cancer and bladder cancer Down-regulation of ncRAN contributes to cell
migration and invasion
BANCR, BRAF-activated non-protein coding RNA Down-regulation of BANCR promotes metastasis
in NSCLC
8 lncRNAs up-regulated
PCA3 (oncogene)
MIR31HG (114-fold upregulated)
8 lncRNAs down-regulated
LINC00261 and PTCSC1 (tumor-suppressor genes)
lncRNA Cancer PathwayFinder
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An lncRNA expression panel can distinguish thyroid cancer
TPR FPR SPC PPV NPV ACC AUC16-gene 1.00 0.08 0.92 0.90 1.00 0.96 0.99
An unsupervised hierarchical clustering with selected lncRNAs showed expression discrimination pattern of the known groups.
Class probability plot when using a16-gene signature panel. Red bar: benign; Blue bar: cancer. X: class probability, >0.5 means classified as cancer.
Cross-verification of classifier
HAND2-AS1 LINC00963 SNHG16 NBR2 PTCSC1 RMRP MALAT1 HEIH
TUG1 LINC00261 CAHM BANCR LINC00538 LINC00312 IPW XIST
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Can integration of mRNA, miRNA and ncRNA data improve signatures?
MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity.Deng Y, Ai J, Guan X, Wang Z, Yan B, Zhang D, Liu C, Wilbanks MS, Escalon BL, Meyers SA, Yang MQ, Perkins EJ.
Integrating microRNA and mRNA expression profiles in response to radiation-induced injury in rat lung.Xie L, Zhou J, Zhang S, Chen Q, Lai R, Ding W, Song C, Meng X, Wu J.
Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.Cava C, Bertoli G, Ripamonti M, Mauri G, Zoppis I, Della Rosa PA, Gilardi MC, Castiglioni I.
Screening biomarkers of prostate cancer by integrating microRNA and mRNA signatures.Feng J, Huang C, Diao X, Fan M, Wang P, Xiao Y, Zhong X, Wu R.
A Systems Biology Approach to Characterize Biomarkers for Blood Stasis Syndrome of Unstable Angina Patients by Integrating MicroRNA and Messenger RNA Expression Profiling.Wang J, Yu G.
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Ingenuity Pathway Analysis (IPA)
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Asking “what’s next?” by modeling, analyzing, and understanding complex 'omics data
Integrate gene expression/miRNA/genomics data Identification of upstream regulators, downstream targets Insight into molecular interactions and cellular phenotypes Discoveries about disease processes 5 million relationships in IPA database
http://www.ingenuity.com/products/ipa
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Understanding miRNA regulation of mRNAs in thyroid carcinoma
microRNA Target Filter: Searched for mRNA targets that were differentially expressed in papillary thyroid carcinoma (PTC) and follicular variant of papillary thyroid carcinoma (FVPTC):
Example: 7 aberrantly expressed miRNAs known to target 7 mRNAs
Filters employed: Inverse relationship between miRNA & mRNA expression Relationships reported in Cancer and/or Endocrine Disorders Thyroid gland expression and selected signaling pathways
http://www.ingenuity.com/products/ipa
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Differentially expressed miRNAs & mRNAs affect invasiveness
Importance of miRNA regulation is shown here by targeting genes involved in invasion of cells necessary for tumor progression and metastasis
http://www.ingenuity.com/products/ipa
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miRNA-mRNA-lncRNA expression patterns affect metastasis progression
Using MAP (Molecule Activity Predictor): Metastasis is predicted to be increased based on observed expression pattern of miRNA, mRNA and lncRNA
http://www.ingenuity.com/products/ipa
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Total RNA Discovery with QIAGEN Sample to Insight Workflows
Complex interaction of coding and non-coding RNAs
Engaging in Total RNA Discovery can result in a deeper understanding of the functional relationships of different RNA molecules
QIAGEN offers Sample to Insight solutions for Total RNA Discovery
Sample prep: any and all samples
Real-time PCR assays and arrays: miRNA, mRNA, lncRNA
Primary data analysis: online tools
Interpretation: Ingenuity Systems IPA
Choose QIAGEN and turn your hypotheses into actionable insights!
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