RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression. Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.

1. Are there biomarkers in
the blood for the type
of autism I study?
QIAGEN kits can help me
collect, stabilize and
isolate RNA from my
blood sample.
I‘ll use the
QIAseq RNA Library
Prep Kit and then run my
samples on an NGS
instrument. This will show
me the RNA levels in
the blood sample.
The Biomedical
Genomics Workbench
shows me that 21 key
genes have markedly
lower levels in autism
patients than in my
control patients.
The IPA software
shows that these genes are
involved in behavioral
disorders, such as autism.
NGS has identified
21 genes that change in
autism. Two techniques are
needed to confirm a result
like this, so I‘ll verify my
data with qPCR.
Do these 21 RNAs that
I identified with NGS
also change when I
look at them by
qPCR?
I can use the same RNA
that I isolated for RNAseq —
it‘s best to test RNA from
the same sample for
consistent results.
I built a custom RT2
Profiler
PCR Array with the GeneGlobe®
Custom Builder tool — this will
let me look at only the RNAs
that interest me.
IPA tells me
exactly which pathways
these RNAs belong to − some
are involved in cell death and
others in neurological development.
GeneGlobe‘s Data
Analysis Portal shows me
that 14 of the original 21
RNAs change in the same
direction with qPCR as
they do in RNAseq.
These 14 RNAs
could be a reliable
blood biomarker
signature, but I need
to validate in a larger
study. I‘m going to
need some help
with a study
of this size.
I‘ve gathered blood samples from 200
autism patients and 200 controls.
Now I can send them to QIAGEN‘s
Service Core and have them check
for my 14 RNAs by qPCR to see
if this biomarker signature
is valid.
My results
from the Service
Core are promising!
The Biomedical
Genomics Workbench
indicates that the RNA
signature is present in
94.6% of the autism
samples.
Time to see if I can
get this into a
clinical trial!
She chooses to use RNA sequencing to look at all the RNAs in her sample. QIAGEN’s NGS
solutions let her perform RNAseq using whatever NGS instrument she has in her facility. The
QIAseq RNA Library Prep Kit generates a library of all the RNAs present in her sample.
Having discovered by RNA sequencing that 21 genes are altered in autism, a new question
arises. How can she verify that the changes in RNA are real – analysis with different techniques
sometimes yields different results. Fortunately, QIAGEN has a solution – our qPCR portfolio.
Sample to Insight:
RNA samples
Sample
collection
stabilization
isolation
Targeted
library
construction
NGS
run
Data
analysis
Interpretation
QIAGEN Webinar Program: “RNA Biomarker Development: Sample to Insight“
PAXgene Blood RNA Kit miRNeasy Serum/Plasma Kit
QIAseq rRNA Depletion Nano Kit RNeasy MinElute Cleanup Kit
QIAseq RNA Library Prep Kit QIAseq Size Selection Kit QIAseq Library Quant System
Biomedical Genomics Workbench Ingenuity Pathway Analysis Software
Ingenuity Pathway Analysis Software
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Webinar Program: “Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR)“
“Critical Factors for Successful Real-Time Multiplex qPCR“
You can use qPCR to verify your sequencing results; RNAseq and qPCR are an increasingly common
combination for discovery and verification in RNA research, including: mRNA, miRNA and lncRNA. Do
the 21 miRNAs identified by RNAseq show the same changes by qPCR analysis?
Biomarker Discovery via RNAseq
Verification via qPCR
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Webinar Program: “Fundamental Concepts and Special Considerations in RNA Isolation“
“Biomarker Discovery, from Sample to Biomarker“
miRNeasy Serum/Plasma Kit
You can use the same isolated RNA from your original blood samples to make sure that you‘re looking at
the same RNA profile. miRNeasy captures all RNA >18 nt, giving you the flexibility to study multiple RNA
species.
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
RT2
Profiler PCR Array RT2
lncRNA PCR Array RT2
SYBR®
Green ROX qPCR Mastermix
RT2
First Strand Kit miScript miRNA PCR Array GeneGlobe Custom Builder Tool
QIAGEN provides customizable qPCR arrays for mRNA, miRNA and lncRNA – just send a list of unique
RNAs that you want to test, and we send you back a pre-made array. It‘s easy with our GeneGlobe
Custom Builder. Here we‘re building a custom RT2
Profiler PCR Array for mRNA.
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Webinar Program: “PCR Array Data Analysis Tutorial“
GeneGlobe Data Analysis Portal Ingenuity Pathway Analysis Software
You can then get insight from your qPCR array results with GeneGlobe’s Data Analysis Portal. Then,
confirm with the same software you used earlier, Ingenuity Pathway Analysis, that the same pathway is
affected. Here, 14 of the original RNAs are verified, and revealed further insight into their roles in cell
death and neurological development.
Larger Studies via QIAGEN‘s Service Core
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Webinar Program: “QIAGEN Service Solutions for Biomarker Research“
GeneGlobe Data Analysis Portal
Again, the answer to one question leads into another. You have a biomarker signature of 14 RNAs that
works well in the samples you‘ve tested. But if this signature can ever make the jump to clinical use, you
need confirmation that it works in a much larger cohort. The first step is to expand your sample size and
repeat the testing. If your lab isn’t big enough to handle such a large number of samples, you can get help
from QIAGEN’s Service Core.
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Service Core
When life science biomarker research leads to a promising result in a large group of samples, the next
step can be to move this research into a clinical trial, where it takes its first steps toward being used for
diagnosis. This is the ultimate goal of much of “translational” or “bench-to-bedside” research – to discover
something that can be turned into a tool for clinicians and used in molecular diagnostics.
Sample
collection
stabilization
isolation
cDNA
synthesis
PCR
run
Data
analysis
Interpretation
QIAGEN Service Core
Researchers in the life sciences face many decision points. Should you study a particular molecule? Should
you use a certain technique? Here, there were multiple points at which we were required to choose a
certain path. The path selected, is highlighted in blue. We could have used biofluids, cells or tissue as our
starting sample. After looking at messenger RNA, we could have examined regulatory mechanisms like
microRNA or long non-coding RNA, or perhaps DNA methylation. You have many avenues to explore to
fully understand why a phenomenon is happening, and a full study can take years and hundreds of
individual experiments to complete. Going down the wrong path could cost considerable time and money.
This is where QIAGEN comes in – we help our life sciences customers at every stage of these multi-stage,
multi-year studies, not only with products, but also with advice and content that can help you make the
right decisions.
Sample to Insight
Trademarks: Ingenuity®
, GeneGlobe®
, QIAGEN®
, Sample to Insight®
, (QIAGEN Group).
SYBR®
(Life Technologies Corporation), PAXgene®
(PreAnalytix GmbH).
© 2016 QIAGEN, all rights reserved
Sample
collection
stabilization
isolation
Targeted
library
construction
NGS
run
Data
analysis
Interpretation
Sample
collection
stabilization
isolation
Targeted
library
construction
NGS
run
Data
analysis
Interpretation
Sample
collection
stabilization
isolation
Targeted
library
construction
NGS
run
Data
analysis
Interpretation
Sample
collection
stabilization
isolation
Targeted
library
construction
NGS
run
Data
analysis
Interpretation
Every life sciences study begins with a question. For example, put yourself in the shoes of this autism
researcher who is watching a webinar about biomarkers in the blood. It sparks your curiosity − could you
find biomarkers like these in your study?
The first step is to stabilize the blood, such as with the PAXgene®
Blood RNA Kit then isolate with the
miRNeasy Serum/Plasma Kit.
You might then choose to use RNA sequencing to look at all the RNAs in your sample. QIAGEN’s NGS
solutions let you perform RNAseq using whatever NGS instrument you have in your facility. The QIAseq
RNA Library Prep Kit generates a library of all the RNAs present in your sample.
Once you‘ve collected your data, you can then generate insights from it using bioinformatics solutions like
the Biomedical Genomics Workbench and Ingenuity®
Pathway Analysis. Here, 21 genes are lower in
autism patients, and IPA confirms the involvement of these genes in certain behavioral disorders.
Having discovered by RNA sequencing that 21 genes are altered in autism, a new question arises. How
can you verify that the changes in RNA are real? Analysis with different techniques sometimes yields
different results. Fortunately, QIAGEN has a solution – our qPCR portfolio.
RNA profiling is a powerful technique for understanding cellular origins
and disease states. Recent studies in a variety of diseases have revealed
RNA signatures that are excellent biomarker candidates for understanding
disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in
discovering a biomarker when you start from a blood sample? Here is the
story of a researcher who is trying to find blood-based biomarkers for
autism spectrum disorders.
So you‘ve identified a set of genes that are differentially expressed in
autism. What‘s your next move? Verification by the gold standard,
quantitative RT-PCR. Let‘s explore the next steps together.
Frequently, your next step in biomarker discovery will be to test your
signature in a larger cohort. When you need more hands to handle such
a large sample size, QIAGEN‘s Service Core can help. Our experts
isolate nucleic acids, run sequencing and qPCR experiments, and even
help with data analysis. Let‘s see how it works.
Blood
sample
RNA
(mRNA)
Identify
potential RNA
biomarkers
by DA
Autism-
focused
targeted
RNAseq
Autism-
focused
qPCR
array
Total
RNAseq
Verify by
a custom
qPCR
array
qPCR
verification
with more
samples
Clinical
study—
diagnostic
marker
Targeted
RNAseq
DNA
methylation
cause mRNA
differences?
DNA
methylation
analysis by
Pyroseq
miRNA
and lncRNA
cause mRNA
differences?
miRNA
and lncRNA
analysis via
RNAseq
Verify
levels by
qPCR
Verify effect
on mRNA
w/ functional
studies
DNA
Identify
genetic
variants by
DA
Whole
genome
sequencing
Targeted
DNA
sequencing
Whole
exome
sequencing
Verify
by qPCR
qPCR
verification
with more
samples
Disease-
variant
association
study
Functional
studies
Drug
development
Clinical
study—
diagnostic
marker
Life Science Researcher: Decision Tree
Solid blue circles represent the workflow choices made in the above example.