Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1

Sample to Insight

Biofluid miRNA profiling: from sample to biomarker

Jonathan Shaffer, Ph.D., Senior Scientist, Product Development

[email protected]

Sample to Insight

Welcome to our four-part webinar series on miRNAs

2

Part 1: Biofluid miRNA profiling: from sample to biomarker

Part 2: Meeting the challenges of miRNA research

Part 3: Advanced miRNA expression analysis

Part 4: Functional analysis of miRNA

miRNA and its role in human disease

Biomarker Discovery in Biofluids: From Sample to Biomarker

Sample to Insight

Biomarker Discovery in Biofluids: From Sample to Biomarker 3

Legal disclaimer

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.

Sample to Insight

QIAGEN tools for biomarker discovery4

Agenda

4

Background1

What is your biomarker I.Q.?2

Sample miRNA biomarker project3

Sample to Insight


What is a biomarker?

A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic responses to a therapeutic intervention

Characteristic Methodology

Presence of antibodies ELISA

Abnormal bp, blood cell counts, electrolyte blood counts, pressure

Distinct histological indicators microscopy

Abnormal liver function markers biofluid assay

Presence of muscle injury protein markers biofluid assay

Elevated kidney marker: serum creatinine biofluid assay

Gene status or gene expression status qPCR, NGS, array, etc.

Sample to Insight


Cancer biomarker

Personalized medicine

Is this the optimal drug

for my cancer?

Predictive

Is it likely to develop this

cancer?

Prognostic

What type of cancer is it?

Diagnostic

What’s the optimal dose

for my body?

Pharmacodynamics

Will the cancer return?

Recurrence

Sample to Insight


Noninvasive biomarker

In order to use a biomarker for diagnostics, the sample must be as easy to obtain as possible

Urine or saliva sample

A drop of blood like those diabetes patients extract

Blood sample taken by a doctor

CSF

Surgical biopsy

Evaluation

Miranda Hanson

Need to list source for image.

Sample to Insight


Canonical pathway of miRNA 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 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

Sample to Insight


Why miRNA biomarkers?

Changes in miRNA can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging and disease

miRNA expression analysis is the foundation for these discoveries

miRBase Entries

Sample to Insight


Circulating miRNA biomarkers in the press

No shortage of candidate genes

Cho (2011) Front. Gene. 2

MicroRNA Deregulation in cancer Theragnostic and prognostic value Reference

let-7a Decrease in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)

let-7f Decrease in NSCLC Associated with overall survival in NSCLC Silva et al. (2011)

miR-1 Decrease in NSCLC Associated with overall survival in NSCLC Hu et al. (2010)

miR-10b Increase in breast cancer Associated with metastases in breast cancer Roth et al. (2010)

miR-17 Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)

miR-17 + 106a Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)

miR-17-3p Increase in CRC Discriminate CRC from healthy controls Ng et al. (2009)

miR-17-5p Increase in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)

miR-20b Decrease in NSCLC Associated with advanced stages and lymph node metastases in NSCLC Silva et al. (2011)

miR-21Increase in CLL harboring 17p deletion

Increase in gastric cancer

Associated with overall survival in CLL

Discriminate gastric cancer from healthy controls

Rossi et al. (2010)

Tsujiura et al. (2010)

miRs-21 + 126 + 210 + 486-5p Deregulate in NSCLC Discriminate stage I NSCLC from healthy controls Shen et al. (2011)

miRs-21 + 155 + 196a + 210 Increase in pancreatic adenocarcinoma Discriminate pancreatic adenocarcinoma from healthy controls Wang et al. (2009)

Sample to Insight


Analytes:

Cell-free miRNA, mRNA, DNA

Samples:

Serum, plasma,cerebrospinal fluid (CSF)

Sample to Insight


What is blood?

RBC, WBC, platelets, CTC, “other cells”, extracellular?

RBC, WBC,platelets, other cells (e.g., circulating tumor cells)

Serum (post clotting)

Plasma (no-clotting)

High levels of nucleases present in plasma Freely circulating nucleic acids should be rapidly degraded Surprisingly, stable nucleic acids can be detected in serum and plasma

Sample to Insight


Stable miRNAs in circulation

An evolving story

1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-659

2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e36943) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-174524) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc. Natl. Acad.

Sci 108: 5003-50085) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:4236) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res.

2010 Nov 1;38(20):7248-59.

Sample to Insight


Exosomes / microvesicles (MVs)

It’s the cargo that matters

Blood Plasma

MVs/exosomes are ~50–200 nm small vesicles excreted by all cells

MVs/exosomes are found in all biofluids (e.g., blood)

MVs/exosomes contain stable RNA (mRNA, miRNA, other small RNAs), DNA and protein

Contents are specifically packaged

Mechanism of local and distant cellular communication

Promise for disease detection and monitoring

Use exosomal miRNA profiling in the absence of tissue to accurately reflect the tumor’s profile

Sample to Insight


Should you only look at exosomes?

No … well, sometimes!

Potentially 90% of miRNAs in circulation are present in a non-membrane-bound form consistent with a ribonucleoprotein complex

At the same time, analyzing the exosome fraction could maximize signal-to-noise ratio of a potential biomarker, providing better sensitivity

Sample to Insight


Agenda

16

Background1



Isolationa

Quantificationb

Sample to Insight


PAXgene® Blood miRNA System

Collect / stabilize and purify total RNA from whole blood

Collection/Stabilization: PAXgene Blood RNA Tubes Draw volume: 2.5 ml Preservation of cellular RNA at point of collection Sample stabilization during transport and storage

Up to 3 days at 18–25°C Up to 5 days at 2–8°C Seven years storage at –20°C or –80°C (studies ongoing)

Purification: PAXgene Blood miRNA Kit High purity RNA from PAXgene-stabilized whole blood High yields of small RNA species Manual and automated solutions

Sample to Insight


Total RNA (miRNA + mRNA) isolation from cell-free body fluids

miRNeasy Serum / Plasma Kit

Includes synthetic RNA control assay for normalization

Minimal elution volume (14 µl)

High-purity RNA suitable for all downstream applications

Easy, robust procedures

Automatable protocol

When possible, avoid heparanized plasma If you can‘t avoid heparinized samples, let me know! We

have a solution!

Purification of circulating RNA from plasma, serum, CSF, saliva, urine, etc.

Sample to Insight


Exosome purification and total RNA isolation from serum / plasma

exoRNeasy Serum/Plasma Maxi KitexoRNeasy Serum/Plasma Midi Kit

Specifically enriches for RNA contained in vesicles

Quickly isolates purified total RNA from microvesicles

Efficiently isolates mRNA and miRNA from plasma/serum

Enables use of high input volumes for sensitive detection of low abundance transcripts Maxi: 4 ml Midi: 1 ml

Sample to Insight


Intact vesicles are eluted from the exoEasy column

Scanning EM (20000x magnification) reveals higher purity with exoEasy

Both preparations contain vesicle-shaped structures within an expected size range UC: Many smaller, unidentified structures/particles that do not match the expected size exoEasy: Intact vesicles with higher purity

Ultracentrifugation (UC) Eluate from exoEasy

Sample to Insight


exoRNeasy Serum / Plasma Maxi Kit

Workflow

Microvesicle isolation 20 minutes

RNA isolation 35 minutes

Separate serum / plasma

Isolate exosomes

Isolate RNA

Sample to Insight


Agenda

22

Background1



Isolationa

Quantificationb

Sample to Insight


Three phase workflow: sample to classifier

Phase I: Qualitatively determine expressed miRNAsPooled samples

miRNome profiling (or NGS)

Phase II: Quantitatively determine differentially expressed miRNAsIndividual profiling of samples (that went into pools)

Screen only expressed miRNAs from Phase I

Phase III: Develop and test classifier modelAddition individual profiling of samples

Screen differentially expressed miRNAs from Phase II

Sample to Insight


miScript PCR System

Complete miRNA quantification system

1. miScript II RT Kit HiFlex Buffer: Unparalleled flexibility for miRNA and

mRNA quantification from a single cDNA preparation HiSpec Buffer: Unmatched specificity for mature

miRNA profiling

2. miScript miRNA PCR Arrays miRNome Pathway-focused

3. miScript PreAMP Kit Optional step for small or precious samples Full miRNome profiling from as little as 1 ng RNA

4. Assays miScript Primer Assays miScript Precursor Assays QuantiTect Primer Assays

5. miScript SYBR Green PCR Kit QuantiTect SYBR Green PCR Master Mix Universal Primer

6. miScript miRNA PCR Array data analysis software Straightforward, free data analysis

Sample to Insight


miScript PCR System

Workflow

1. Isolate total RNA

2. Perform reverse transcription

3. Prepare PCR pre-mix

4. Load PCR arrays or plates

5. Perform real-time PCR

6. Analyze data

Sample to Insight


miRNA expression profiling: miScript miRNA PCR Arrays

Wet-lab-verified miScript Primer Assays pre-dried in PCR plates

miRBase Profiler miRNome Arrays Most species Largest content

High content (HC) arrays Targeted miRNome profiling

Focused arrays Bioinformatic-driven profiling

Formats 96-well, 384-well, Fluidigm® BioMarkTM

Compatible with virtually all mainstream real-time instruments Fully customizable

Prep your PCR reaction mix Load your plate Run your real-time experiment!

No pipetting of individual primers!

Sample to Insight


miRNA expression profiling: miScript miRNA PCR Arrays (cont.)

miRBase Profiler miScript miRNA PCR Array

Human Coverage through miRBase v21 2402 primer assays!

Mouse Coverage through miRBase v21 1765 primer assays!

Rat Dog Rhesus macaque Cow

100% validated assays Each assay is bench validated Each array is quality controlled

Leading miRNome coverage

Completely scalable! Choose as many plates as you

want … profile the v21 miRNome … profile only the v16 miRNome

Contact product development if there is interest in other species!

miRBase Profiler Arrays Benefits of miRBase Profiler Arrays

The most complete, validated miRNome available!

Sample to Insight


Are newly annotated miRNAs even expressed?

Liver tissue profiling of a pool of ten healthy male liver tissues

miScript

Vendor 2

Vendor 3

Sample to Insight


Limiting samples: miScript PreAMP Kit

miRNome profiling from as little as 1 ng total RNA

Highly multiplex, PCR-based preamplification Compatible with all miScript miRNA PCR Arrays and miScript Primer Assays Enables miRNA profiling experiments using very limited amounts of starting material

Cell or tissues: 1 ng total RNA Fluids:

Serum / plasma: 50 µl or less Urine: Any amount CSF: Any amount Aqueous humor: Any amount When in doubt, ‘miScript PreAMP’ it!

Sample to Insight


High-throughput miRNA expression profiling: miScript Microfluidics

First complete system for miRNA expression profiling on the Fluidigm® BioMarkTM

Why use miScript on the BioMark?

96 samples, 384 assays

4 Fluidigm Real-Time PCR Chips

5 h per Chip

36,864 data points in 20 h (only 2 days!)

Sample to Insight


Agenda

31

Background1



Sample to Insight


Liver toxicity miRNA biomarker project with Dr. James Dear

Biomarker Discovery Workflow Acetaminophen (APAP) overdose is a common poisoning worldwide and can cause liver damage, potentially resulting in acute liver failure and death In the US and UK, acetaminophen overdose is the most

common cause of acute liver failure

Scope of collaboration: Determine miRNA markers of acetaminophen poisoning Distinguish acetaminophen poisoning from other liver

syndromes

Experiment workflow: biomarker discovery Phase 1: 356 expressed miRNAs

Narrowed list from 1809 miRNAs

Phase 2: 85 differentially expressed miRNAs

Phase 3: Classifier developed and tested that can separate APAP-TOX from APAP-No TOX

Sample to Insight


Phase I: Qualitatively determine expressed miRNAs

Goal: Establish the “APAP miRNome” by assaying pooled samples with the human miRNome

Samples: 54 acetaminophen (APAP) overdose plasma samples 27 APAP-no TOX: acetaminophen overdose without toxicity 27 APAP-TOX: acetaminophen toxicity

Total RNA isolation: miRNeasy Serum/Plasma KitRandom RNA pool preparation

2 APAP-no TOX pools: 9 samples per pool 2 APAP-TOX pools: 9 samples per pool

miRNA expression profiling: miScript PCR System Human miRNome V18 (1809 bench-verified primers)

Selection: Qualitative determination CT < limit of detection (CT = 35) with a single, sharp melt peak

How many miRNAs were selected? 356

miRNAs expressed in all 4 pools: 132 miRNAs expressed in 3 pools: 105 miRNAs expressed in 2 pools: 114 miRNAs expressed in 1 pool: 5

Sample to Insight


Phase II: Quantitatively determine differentially expressed miRNAs

Goal: Determine miRNAs differentially expressed in response to APAP toxicity

Total RNA Samples: 54 acetaminophen (APAP) overdose plasma samples from Phase I 27 APAP-no TOX: acetaminophen overdose without toxicity 27 APAP-TOX: acetaminophen toxicity

miRNA expression profiling: miScript PCR System 356 human miRNAs Fluidigm BioMark HD (4 runs)

Results: 85 differentially expressed miRNAs

± 3-fold, p-value < 0.05 Well expressed in APAP-no TOX,

APAP-TOX, or both 1 snoRNA 7 invariantly expressed miRNAs

Identified using NormFinder Very important for data normalization

miR-122-5p

miR-885-5p

Sample to Insight


Phase III: Develop and test classifier model

Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX

Step 1: Select the most predictive 16 miRNAs from training data set (93 targets) Train random forest classifier: 250 bootstrap subsamples (80%) without replacement

Determined importance rank of each target Performed prediction on remaining 20% of samples

Class separation magnitude and model error 250 iterations of classifier training: Determined median importance rank of each target

Selected the 16 most predictive targets

Step 2: Train a final random forest classifier using 16 most predictive miRNAs

Most predictive miRNAs

Importance Rank miRNA ID

1 - 11 miRNA #1 - #11

12 hsa-miR-122-5p13 miRNA #13

14 hsa-miR-885-5p15 miRNA #15

16 miRNA #16

Class Separation

Sample to Insight


Phase III: Develop and test classifier model (cont.)

Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX

Step 3: Test the random forest classifier on an independent, blinded test data set (81 samples)

Sensitivity: 90%

Specificity: 92%

Classifier can efficiently separate APAP-no TOX from APAP-TOX

Sample to Insight


Biomarker development

Important considerations

Be novel! Initial whole miRNome screening = unique signatures!

If the group would have only profiled the first two plates (roughly 768 mature miRNAs): Only 64 of 92 differentially expressed or invariant miRNAs would have been assayed

30% loss of data Two invariant miRNAs would have been missed

Five miRNAs from optimal 16 miRNA signature would not have been assayed Three of top five miRNAs from the signature would not have been assayed

CT value normalization is critical! snoRNAs / snRNAs do not exhibit robust expression in cell-free biofluids and should not be

selected as normalization controls Large assay panel normalization: CT mean of commonly expressed miRNAs Small assay panel normalization: Invariant miRNA(s)

Verify miRNA signature on naïve samples Strong changes might be general indicators or even non-specific

Other liver disease? Stress markers? Relatively weak changes might add specificity What’s next?

Revisit literature, Ingenuity® Pathway Analysis (IPA), etc. Screen other types of samples to help define specificity and refine signature.

Sample to Insight


Circulating mRNA Biomarker Discovery

Exosomes contain stable RNA including mRNA Isolation

exoRNeasy Serum / Plasma Maxi Kit Reverse Transcription/PreAMP

RT2 PreAMP cDNA Synthesis Kit RT2 PreAMP Pathway Primer Mix

Real-time PCR RT2 Profiler PCR Arrays RT2 qPCR Primer Assays

Sample to Insight


Agenda

39

Background1



Sample to Insight


Where can I find the products discussed today?

www.qiagen.com

www.qiagen.com/GeneGlobe

Sample to Insight


Sample to Insight miRNA portfolio

miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT Kit HiPerFect Transfection Reagent

miRNeasy 96 Kit miScript Plant RT Kit Attractene Transfection Reagent

miRNeasy FFPE Kit miScript PreAMP Kit miScript miRNA Mimics

miRNeasy Serum/Plasma Kit miScript SYBR Green PCR Kit miScript miRNA Inhibitors

Modified miRNeasy Mini Kit for plant tissues miScript miRNA PCR Arrays Custom miScript miRNA Mimics

PAXgene Tissue miRNA Kit miScript Microfluidics for Fluidigm Mimic and inhibitor controls

PAXgene Blood miRNA Kit miScript Primer Assay miScript Target Protector

Supplementary protocol for miRNA from Plasma and Serum miScript Precursor Assay miScript miRNA Inhibitor 96 and 384

Plates and Sets

Prof

iling

QIAcube QIAgility Rotor-Gene QQIAGEN Service Core

FunctionalizationIsolation Quantification and profiling

Sample to Insight


Sample to Insight mRNA portfolio

Isolation Quantification and profiling

RNeasy Mini Kit RT2 First Strand cDNA Kits

RNeasy Microarray Tissue Mini Kit RT2 qPCR Master Mixes

RNeasy FFPE Kit RT2 Profiler PCR Arrays (Profiling)

RNeasy Micro Kit RT2 qPCR Primer Assays

PAXgene Blood RNA Kit GeneGlobe Data Analysis Center

QIAcube QIAgility Rotor-Gene QHigh-throughput

Sample to Insight


Thank you for attending today’s webinar!

Jonathan Shaffer, [email protected]

Contact [email protected]

Questions?

Healthcare

Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1