Circulating miRNAs have great potential as biomarkers due to their aberrant expression in cancer and other diseases. However, miRNAs from body fluids are hard to obtain in amounts sufficient for detailed miRNome profiling. This slideshow describes an integrated, PCR-based system that reduces the amount of sample required for full miRNome profiling by several orders of magnitude and provides unparalleled reproducibility and precision. Detailed protocols are highlighted regarding RNA isolation, real-time quantification and data analysis for the assessment of serum, plasma, urine and cerebrospinal fluid samples. This system enables accurate miRNA analysis on the smallest of samples and opens up new possibilities for biomarker development.
Dehradun Call Girls Service 08854095900 Real Russian Girls Looking Models
Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1
1. Sample to Insight
Biofluid miRNA profiling: from sample to biomarker
Jonathan Shaffer, Ph.D., Senior Scientist, Product Development
Jonathan.Shaffer@qiagen.com
2. 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
3. Sample to Insight
Legal disclaimer
Biomarker Discovery in Biofluids: From Sample to Biomarker 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.
4. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
4
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
5. Sample to Insight
What is a biomarker?
Biomarker Discovery in Biofluids: From Sample to Biomarker 5
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.
6. Sample to Insight
Cancer biomarker
Biomarker Discovery in Biofluids: From Sample to Biomarker 6
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
7. Sample to Insight
Noninvasive biomarker
Biomarker Discovery in Biofluids: From Sample to Biomarker 7
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
8. Sample to Insight
Canonical pathway of miRNA biogenesis
Biomarker Discovery in Biofluids: From Sample to Biomarker 8
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
9. Sample to Insight
Why miRNA biomarkers?
Biomarker Discovery in Biofluids: From Sample to Biomarker 9
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
10. Sample to Insight
Circulating miRNA biomarkers in the press
Biomarker Discovery in Biofluids: From Sample to Biomarker 10
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-21
Increase 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)
11. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 11
Analytes:
Cell-free miRNA,
mRNA, DNA
Samples:
Serum, plasma,
cerebrospinal fluid (CSF)
12. Sample to Insight
What is blood?
Biomarker Discovery in Biofluids: From Sample to Biomarker 12
RBC, WBC, platelets, CTC, “othercells”,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
13. Sample to Insight
Stable miRNAs in circulation
Biomarker Discovery in Biofluids: From Sample to Biomarker 13
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:e3694
3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-17452
4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc. Natl. Acad.
Sci 108: 5003-5008
5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423
6) 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.
14. Sample to Insight
Exosomes / microvesicles (MVs)
Biomarker Discovery in Biofluids: From Sample to Biomarker 14
It’sthecargothatmatters
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 reflectthetumor’s
profile
15. Sample to Insight
Should you only look at exosomes?
Biomarker Discovery in Biofluids: From Sample to Biomarker 15
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
16. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
16
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
Isolationa
Quantificationb
17. Sample to Insight
PAXgene® Blood miRNA System
Biomarker Discovery in Biofluids: From Sample to Biomarker 17
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
18. Sample to Insight
Total RNA (miRNA + mRNA) isolation from cell-free body fluids
Biomarker Discovery in Biofluids: From Sample to Biomarker 18
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.
19. Sample to Insight
Exosome purification and total RNA isolation from serum / plasma
Biomarker Discovery in Biofluids: From Sample to Biomarker 19
exoRNeasy Serum/Plasma Maxi Kit
exoRNeasy 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
20. Sample to Insight
Intact vesicles are eluted from the exoEasy column
Biomarker Discovery in Biofluids: From Sample to Biomarker 20
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
21. Sample to Insight
exoRNeasy Serum / Plasma Maxi Kit
Biomarker Discovery in Biofluids: From Sample to Biomarker 21
Workflow
Microvesicle
isolation
20 minutes
RNA isolation
35 minutes
Separate serum /
plasma
Isolate
exosomes
Isolate
RNA
22. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
22
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
Isolationa
Quantificationb
23. Sample to Insight
Three phase workflow: sample to classifier
Biomarker Discovery in Biofluids: From Sample to Biomarker 23
Phase I: Qualitatively determine expressed miRNAs
Pooled samples
miRNome profiling (or NGS)
Phase II: Quantitatively determine differentially expressed miRNAs
Individual profiling of samples (that went into pools)
Screen only expressed miRNAs from Phase I
Phase III: Develop and test classifier model
Addition individual profiling of samples
Screen differentially expressed miRNAs from Phase II
24. Sample to Insight
miScript PCR System
Biomarker Discovery in Biofluids: From Sample to Biomarker 24
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
25. Sample to Insight
miScript PCR System
Biomarker Discovery in Biofluids: From Sample to Biomarker 25
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
26. Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays
Biomarker Discovery in Biofluids: From Sample to Biomarker 26
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!
27. Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays (cont.)
Biomarker Discovery in Biofluids: From Sample to Biomarker 27
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…profilethe v21 miRNome
…profileonly 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!
28. Sample to Insight
Are newly annotated miRNAs even expressed?
Biomarker Discovery in Biofluids: From Sample to Biomarker 28
Liver tissue profiling of a pool of ten healthy male liver tissues
miScript
Vendor 2
Vendor 3
29. Sample to Insight
Limiting samples: miScript PreAMP Kit
Biomarker Discovery in Biofluids: From Sample to Biomarker 29
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!
30. Sample to Insight
High-throughput miRNA expression profiling: miScript Microfluidics
Biomarker Discovery in Biofluids: From Sample to Biomarker 30
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!)
31. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
31
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
32. Sample to Insight
Liver toxicity miRNA biomarker project with Dr. James Dear
Biomarker Discovery in Biofluids: From Sample to Biomarker 32
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
33. Sample to Insight
Phase I: Qualitatively determine expressed miRNAs
Biomarker Discovery in Biofluids: From Sample to Biomarker 33
Goal: Establishthe“APAPmiRNome”byassayingpooledsampleswiththehumanmiRNome
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 Kit
Random 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
34. Sample to Insight
Phase II: Quantitatively determine differentially expressed miRNAs
Biomarker Discovery in Biofluids: From Sample to Biomarker 34
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
35. Sample to Insight
Phase III: Develop and test classifier model
Biomarker Discovery in Biofluids: From Sample to Biomarker 35
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-122-5p
13 miRNA #13
14 hsa-miR-885-5p
15 miRNA #15
16 miRNA #16
Class Separation
36. Sample to Insight
Phase III: Develop and test classifier model (cont.)
Biomarker Discovery in Biofluids: From Sample to Biomarker 36
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
37. Sample to Insight
Biomarker development
Biomarker Discovery in Biofluids: From Sample to Biomarker 37
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.
38. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 38
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
39. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
39
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
40. Sample to Insight
Where can I find the products discussed today?
Biomarker Discovery in Biofluids: From Sample to Biomarker 40
www.qiagen.com
www.qiagen.com/GeneGlobe
41. Sample to Insight
Sample to Insight miRNA portfolio
Biomarker Discovery in Biofluids: From Sample to Biomarker 41
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
Profiling
QIAcube QIAgility Rotor-Gene QQIAGEN Service Core
FunctionalizationIsolation
Quantification
and profiling
42. Sample to Insight
Sample to Insight mRNA portfolio
Biomarker Discovery in Biofluids: From Sample to Biomarker 42
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
43. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 43
Thank you for attending today’s webinar!
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
Contact QIAGEN
1-800-426-8157
BRCsupport@QIAGEN.com
Questions?
Editor's Notes
PreAnalytiX is using a systems approach offering a collection device as well as a purification kit.
The PAXgene Blood RNA Tube is a plastic tube with 2.5 ml draw volume. RNA is preserved at the time point of collection and can be stored and transported at various temperatures. For shipment issues a huge benefit is the stabilization for up to 3 days at room temperature and up to 5 days at 4°C.
Furthermore, we there are ongoing studies in our laboratories where we could claim for 6-7 years stability at -20°C and -80°C.
High yields of small RNA species. Blood was collected and stored for 20 – 24 hours at room temperature in the PAXgene Blood RNA Tube before freezing at - 15 to - 30°C. RNA was purified using the PAXgene Blood RNA Kit (RNA kit) or the PAXgene Blood miRNA Kit (miRNA kit). (A) Purified RNA was analyzed by agarose gel electrophoresis