Download PrimeFlow™ RNA Assay Technology Validation Paper

PrimeFlow™ RNA Assay
Technology Validation Paper
Introduction
PrimeFlow™ RNA Assay reveals the dynamics of RNA and protein expression within individual cells, facilitating unprecedented analysis of
their correlation as the cells change over time or in response to stimulation. This novel assay uses fluorescent in situ hybridization (FISH) to
enable simultaneous detection of as many as three RNA transcripts in a single cell using a standard flow cytometer. PrimeFlow RNA Assay is
compatible with cell surface and intracellular staining, using common flow cytometry fluorochromes. The assay is based upon proven and
well-published ViewRNA assays designed for microscopic analysis of RNA in cells and tissues that combine paired oligonucleotide probe
design with branched DNA (bDNA) signal amplification to robustly detect gene expression at the single-cell level.
Coupling RNA expression with protein detection on a flow cytometer generates multiparametric data in heterogeneous cell populations
and offers in-depth and high content details at the single-cell level. In contrast, microarrays and sequencing can provide comprehensive
gene expression data in bulk sample preparations; however, the analysis of bulk samples can mask the individual effects of unique
cellular subsets. Using PrimeFlow RNA Assay, specific cell populations may be analyzed for unique transcript expression levels, or cell
subsets evaluated over time to determine transcriptional regulation and protein expression simultaneously. Such unique and valuable
insights are highly applicable to answering previously unanswerable questions and have broad implications for advancing research
across multiple fields of biology.
n
n
n
n
n
Observe the heterogeneity of gene expression at a single-cell level
Correlate RNA and protein kinetics within the same cell
Detect non-coding RNA in cell subsets
Evaluate viral RNA expression in infected cells
Analyze mRNA expression levels when antibody is unavailable
Figure 1: Example data set
Ki-67
RNA vs CD8
Granzyme B
Protein vs CD8
Protein vs RNA
RNA vs CD8
Protein vs CD8
Protein vs RNA
CD8 PE-eFluor® 610
CD8 PE-eFluor® 610
Ki-67 mRNA Alexa Fluor® 488
CD8 PE-eFluor® 610
GrzB Protein PE-Cyanine7
GrzB Protein PE-Cyanine7
GrzB mRNA Alexa Fluor® 750
Ki-67 Protein eFluor® 450
Ki-67 Protein eFluor® 450
Ki-67 mRNA Alexa Fluor® 488
Unstimulated:
CD8 PE-eFluor® 610
GrzB mRNA Alexa Fluor® 750
CD8 PE-eFluor® 610
Ki-67 mRNA Alexa Fluor® 488
GrzB Protein PE-Cyanine7
GrzB Protein PE-Cyanine7
GrzB mRNA Alexa Fluor® 750
CD8 PE-eFluor® 610
Ki-67 Protein eFluor® 450
Ki-67 Protein eFluor® 450
Ki-67 mRNA Alexa Fluor® 488
2-day Anti-CD3/CD28 stimulated:
CD8 PE-eFluor® 610
CD8 PE-eFluor® 610
GrzB mRNA Alexa Fluor® 750
PrimeFlow™ RNA Assay in action
C57Bl/6 splenocytes were unstimulated (top row) or stimulated for 2 days with Anti-Mouse CD3 and CD28 Functional Grade Purified antibodies (cat. no. 16-0031 and
cat. no. 16-0281) (bottom row) and in the presence of Protein Transport Inhibitor Cocktail (cat. no. 00-4980) for the last 3 hours of culture, followed by analysis using
the PrimeFlow™ RNA Assay (cat. no. 88-18001). Cells were fixed and permeabilized using the PrimeFlow™ RNA Assay buffers and protocol, then intracellularly stained with
Anti-Mouse CD8a PE-eFluor ® 610 (cat. no. 61-0081), Anti-Mouse Ki-67 eFluor ® 450 (cat. no. 48-5698), and Anti-Mouse Granzyme B PE-Cyanine7 (cat. no. 25-8898).
Cells were then hybridized with Type 6 Mouse Granzyme B Alexa Fluor ® 750 (cat. no. VB6-16522), Type 4 Mouse Ki-67 Alexa Fluor ® 488 (cat. no. VB4-16518), and
Type 1 Mouse β-actin Alexa Fluor ® 647 (cat. no. VB1-10350) target probes.
Assay technology
Fluorescent in situ hybridization (FISH) is a powerful technique that allows specific localization of ribonucleic acid targets in fixed cells.
The basic premise of the application relies on detecting nucleic acids through sequential hybridization of nucleic acid probes that
provides gene expression information at a single-cell level. Traditional FISH techniques are generally limited by high background and
low sensitivity due to non-specific binding and inefficient signal amplification.
PrimeFlow™ RNA Assay incorporates a proprietary oligonucleotide probe set design and branched DNA (bDNA) signal amplification
technology to analyze RNA transcripts by flow cytometry. bDNA technology provides a unique approach to RNA detection and signal
amplification by amplifying the reporter signal rather than the target sequence (e.g., PCR) for consistent results, a common problem
for PCR-based assays.
Figure 2: Branched DNA (bDNA) probe design principle
Label Probe
Label
Probe
Amplifier
Amplifier
Pre-amplifier
Pre-Amplifier
In the PrimeFlow™ RNA Assay, target-specific probe sets contain
20 to 40 oligonucleotide pairs that hybridize to the target RNA
transcript. Signal amplification is achieved through specific
hybridization of adjacent oligonucleotide pairs to bDNA structures,
formed by Pre-amplifiers, Amplifiers, and fluorochrome-conjugated
Label Probes, resulting in excellent specificity, low background, and
high signal-to-noise ratio (Figure 2).
RNA
RNA
Left Oligo
RightOligo
Oligo
Right
Figure 3: PrimeFlow™Sample
RNA Assay workflow
Sample
Preparation
Sample
Sample
Preparation
Sample
Sample
Preparation
Preparation
Sample
preparation
Preparation
Preparation
(Antibody staining
staining
Label(Antibody
proteins with
(Antibody
staining
optional)
(Antibody
staining
antibody
(optional)
optional)
(Antibody
staining
(Antibody
staining
(Antibody
staining
optional)
optional)
Fix
and
cells
Fix permeabilize
and Permeabilize
optional)
optional)
Fix and Permeabilize
suspension cells
in suspension
Fix and Permeabilize
suspension
cells
FixFix
and
Permeabilize
and
Permeabilize
Fix suspension
and
Permeabilize
cells
Label
intracellular
proteins
suspension
cells
suspension
cells
suspension
cells
with
antibodies
(optional)
Gene 1
Gene 1
Gene
1
2
Gene
Gene
1
Gene
Gene
Gene
1 12
Gene 2
Gene 2
Gene
Gene
2 2
Suspension cells
Suspension cells
with RNA fixed
Suspension
cells
with RNAcells
fixed
Suspension
Suspension
cells
Suspension
cells
with
RNA
fixed
Suspension
cells
with
RNA
fixed
with
RNA
fixed
with
withRNA
fixedfixed
RNA
2
Target
Target
Hybridization
Target
Target
Hybridization
Target
Target
Hybridization
Hybridization
Target
hybridization
Hybridization
Hybridization
Gene-specific
Label
Gene-specific Label
Extenders (LE)
Gene-specific
Label
Extenders (LE)
Gene-specific
Label
Gene-specific
Gene-specific
Label
Gene-specific
Label
Extenders
(LE)
Extenders
(LE)
Label
Extenders
Extenders
(LE)(LE)
Extenders
(LE)
Gene-specific
Gene-specific
Blocking Probes (BL)
Gene-specific
Blocking Probes (BL)
Gene-specific
Gene-specific
Gene-specific
Blocking
Probes
(BL)
Blocking
Probes
(BL)
Blocking
Probes
(BL)
Blocking
Probes
(BL)
Incubate cells with
Incubate cells with
Incubate cells
with
gene-specific
probe
Gene Specific
Incubate
cells
with
Gene
Specific
sets
(Type
1,
4,
or
6)
Incubate
cells
with
probe sets
probe
sets
Incubate
cells with
Incubate
cells
Gene
Specific
probe
sets with
Gene
Specific
(Type
1, 4 or 6)
Gene
Specific
Gene
Specific
probe
sets
(Type
1, 4 or 6)
probe
sets
probe
sets
probe
sets
(Type
1,
4 or
(Type
1,
4
or
6) 6)
(Type
4 or
(Type
1, 41,or
6) 6)
Signal
Signal
Amplification
Signal
Signal
Amplification
Signal
Signal
Amplification
Amplification
Signal
amplification
Amplification
Amplification
PreAmplifier
PreAmplifier
PreAmplifier
PreAmplifier
Pre-amplifier
PreAmplifier
PreAmplifier
Amplifier
Amplifier
Amplifier
Amplifier
Amplifier
Amplifier
Amplifier
Hybridization of
Hybridize
with
Hybridization
of
Pre-Amplifier and
Hybridization
of
Pre-amplifier
andofand
Pre-Amplifier
Hybridization
Amplifier
DNA
Hybridization
Hybridization
of of
Pre-Amplifier
and
Amplifier
Amplifier
DNADNA
Pre-Amplifier
and
(Type 1, 4, and
6)
Pre-Amplifier
and
Pre-Amplifier
and
Amplifier
(Type
4,
and
6)
(Type
1, 4, 1,
orDNA
6)DNA
Amplifier
Amplifier
DNA
Amplifier
DNA
(Type
1,
4,
and
6)
(Type
1,
4,
and
6)
(Type
1, and
4, and
(Type
1, 4,
6) 6)
Detection
Detection
Detection
Detection
Detection
Detection
Detection Label
Fluorescent
Fluorescent Label
Probe
Fluorescent
Label
Probe
Fluorescent
Label
Fluorescence-labeled
probes
Fluorescent
Label
Fluorescent
Probe Label
Probe
Label
Probes
to
Add Add
labeled
probes
to cells
Probe
Probe
Add
Label
Probes to
cells
Add
Label
Probes
cells
Add
Label
Probes
to to
Add
Label
Probes
Add
Label
Probes
to to
cells
cells
cells
cells
process cells using
process cells using
a standard Flow
process
cells
using
a standard
Flow
process
cells
using
Process
using
Cytometer
Instrument
process
cells
using
process
cells
using
a
standard
Flow
Cytometer
Instrument
a standard
Flow
cytometer
aflow
standard
Flow
a astandard
Flow
Cytometer
Instrument
Cytometer
Instrument
Cytometer
Instrument
Cytometer Instrument
CD8 mRNA
AlexaFluor® 647
Left Oligo
CD8 PE-Cyanine7
PrimeFlow™ RNA Assay principle
The assay workflow contains several steps: surface antibody staining; fixation and permeabilization; intracellular antibody staining,
followed by target probe hybridization, with RNA-specific probe sets; signal amplification using bDNA constructs and detection by flow
cytometry. For simplicity, detection of only two RNA targets are shown in orange and yellow (Figure 3, Page 2) with only three of the
20 to 40 oligonucleotide target probe pairs per target RNA.
Antibody staining, fixation, and permeabilization
Single-cell suspensions can be stained for cell surface markers and fixable viability dyes before the cells are fixed and permeabilized.
Subsequently, the cells may be stained with antibody directed to intracellular targets, such as transcription factors and cytokines.
Spacers
After an additional fixation step, the
cells are ready to proceed through the hybridization and signal amplification steps.
mRNA
Target hybridization
Target probes
(pairs)
A target-specific
Probe
Set (”Roots”)
contains 20 to 40 oligonucleotide pairs that hybridize to specific regions across the target RNA sequence.
(minimum
pairs per mRNA)
Subsequent
signal20amplification
requires that each half of a given oligonucleotide pair binds to the target RNA in adjacent positions.
Three types of Probe Sets are currently available to allow detection of RNA labeled with Alexa Fluor® 647 (Type 1 Probe Sets), Alexa Fluor® 488
(Type 4 Probe Sets), or Alexa Fluor® 750 (Type 6 Probe Sets). When detecting more than one RNA target in a single sample, each Probe
Set must be a unique type to differentiate its signal from the others.
Figure 4A: Probe Set hybridization
mRNA
Probe Set
Probe Set Type
Fluorochrome Label
Excitation
Wavelength (max)
Emission
Wavelength (max)
Laser Excitation
Wavelength
Bandpass Filter
Recommendation
Type 1
Alexa Fluor® 647
647 nm
668 nm
632-640 nm
660/20
Type 4
Alexa Fluor® 488
488 nm
519 nm
488 nm
530/30
Type 6
Alexa Fluor 750
749 nm
775 nm
632-640 nm
780/60
®
Signal amplification
Signal amplification using bDNA technology is achieved through a series of sequential hybridization steps, that forms a tree-like
structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes to form the trunk of the tree.
Label Probes (”Leaves”)
(Fluorochrome-conjugated
oligo
Multiple Amplifier molecules hybridize to their respective Pre-amplifier
to create the branches. Finally, multiple Label
Probes hybridize
Amplifier (”Branches”)
Bind to multipe sites on Amp
Bind to multiple sites on PreAmp
to the Amplifiers and form the “leaves” of the tree. A fully assembled signal amplification tree contains 400 Label Probe binding
sites. If all target-specific oligonucleotides in a 20 oligonucleotide pair Probe Set bind to the target RNA transcript, an 8,000-fold
amplification can be achieved.
Fig 3a
Branched DNA Tree Assembly
Figure 4B: Pre-amplifier (“trunk”) binds
to a Probe Set
Figure 4C: Amplifier (“branches”) binds
to multiple sites on Pre-amplifier
Figure 4D: Label Probes (“leaves”)
fluorochrome-conjugated oligo binds
to multiple sites on Amplifier
Fluorescence detection
Upon completion of the assay protocol, target RNA data is detected in cells by analyzing the sample on a standard flow cytometer equipped
with 633-647 nm and 488 nm lasers and appropriate filter configurations, to capture the fluorescent signals (see table in Figure 4A).
3
Precision
Intra-assay variability
To assess intra-assay variability, samples from stimulated and unstimulated human peripheral blood mononuclear cells (PBMC) were
divided into seven tubes and assessed for expression of Ribosomal Protein L13a (RPL13a), a positive control gene expressed in all
PBMC, and interferon gamma (IFNγ), induced only upon stimulation in a subset of lymphocytes. As shown in Figures 5A and 5B, the
assay shows robust intra-assay performance, with a CV% less than 10% for both RPL13a and IFNγ.
Figure 5A
Figure 5B
6000
6000
10.7
1600
1600
RPL13a Alexa Fluor® 750 MedFl
IFNγ Alexa Fluor® 750 MedFl
1800
1800
1400
1400
1200
1200
1000
1000
800
800
600
600
400
400
200
200
5000
5000
8.2
7.3
STIM
UNSTIM
4000
4000
3000
3000
2000
2000
1000
1000
0
00
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Assessing intra-assay variability
A single sample of stimulated or unstimulated human PBMC was divided into 7 tubes and assessed for expression of RPL13a mRNA in total lymphocytes (Figure 5B)
or IFNγ mRNA in IFNγ + events (Figure 5A). Data shown are the average of the 7 replicates, and error bars represent standard deviation. Values shown above the bars
represent the CV%.
To assess the effect of target probe handling on assay variability, a sample of mouse splenocytes was divided into three samples and
assayed for expression of ß-actin, RPL13a, ß2-microglobulin (B2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Peptidylprolyl cis-trans isomerase B (PPIB). Target probes were diluted independently for each triplicate. As shown below, the CV% were typically
less than 10%, while RPL13a was slightly higher, around 12%.
Intra-Experiment Variability by Probe Set
14%
14%
12%
12%
CV%
CV
10%
10%
8%
8%
6%
6%
4%
4%
2%
2%
0%
0%
Actin
Actin Actin
Actin
PPIB
Actin
Actin RPL13aRPL13aRPL13a
RPL13α RPL13α RPL13α B2M
B2M GAPDH
GAPDH
PPIB
Type 11 Type
Type 4
4 Type
Type 66 Type
Type 11 Type
Type 44 Type
Type 56 Type
Type 44 Type
Type 44 Type
Type 11
Type
AF647 AF488 AF647
AF647 AF488
AF488 AF488 AF647
AF647AF488AF750AF647AF488AF750AF488AF488
AF647
AF=Alexa Fluor®
Figure 6: Contribution of target probe dilution to assay variability
Splenocytes from C57Bl/6 mice were assessed for expression of several positive
control genes. Samples were prepared in triplicate with independent dilutions of
target probes made for each sample. CV% of the median fluorescence intensity for
each gene are shown.
4
Operator variability
To assess the contribution of technicians to assay variability, aliquots of U937 cells from the same cell culture were tested in triplicate
for GAPDH, PPIB, and B2M gene expression by two different technicians. No Probe (NP) controls were used as negative controls, and
samples were analyzed using the same cytometer settings. For each operator, the CV% of the mean fluorescence intensity (MFI) of
triplicate samples was less than 6%, consistent with previous data for intra-assay variability. The variation between operators was
approximately 5% for the MFI, and 20% for the signal-to-noise ratio, calculated as the ratio between the positive MFI and the No
Probe control MFI.
Figure 7B
Figure 7A
Operator 1 GAPDH-Type 4
Operator 2
Operator 1
GAPDH-Type 4
Operator 2
500
400
Count
350
300
MFI
Count
Count
Count
Count
450
250
200
PPIB-Type®1
Type 4 GAPDH Alexa Fluor 488
150
PPIB-Type 1
100
50
NP GAPDH NP PPIB
NP B2M NP GAPDH NP PPIB NP B2M
Type 4 Type 4Type 1 Type 1 Type 6 Type 6 Type 4Type 4 Type 1 T ype 1 Type 6Type 6
AF488 AF488AF647AF647 AF750AF750AF488AF488AF647AF647AF750AF750
Count
Count
Count
Count
Count
0
*AF = Alexa Fluor®
Type 1 PPIB Alexa Fluor® 647
B2M-Type6 6
B2M-Type
FL4 red
FL4 red
FL4 red
Count
Count
Count
Count
Count
Count
FL4 red
Type 6 B2M Alexa Fluor® 750
FL5red
red
FL5
FL5red
red
FL5
Contribution of operator variability
U937 cells were assessed for expression of several positive control genes. Samples were prepared in triplicate by two technicians. Histogram overlays of the triplicates for
No Probe and target probes are shown on the left (Figure 7A). MFI for each sample are shown on the right (Figure 7B).
5
Additional studies to assess variation between operators were run with samples containing only a subpopulation of positive cells.
C57Bl/6 splenocytes were stimulated for three days with anti-CD3 and anti-CD28 antibodies, and assessed for expression of Ki-67 and
Granzyme B mRNA. The samples were divided among four technicians who independently performed the assay. As shown below, the
mean percentage of positive events for Ki-67 or Granzyme B were 18.6% and 30.6%, respectively (Figure 8A). The MFI of the mRNA
positive events are shown in the bar graph, and in this case the CV was 12-15% (Figure 8B), consistent with the results obtained with
positive control genes in U937 cells (Figure 7A & 7B).
Figure 8A
Count
Count
Contribution of operator variability in a bimodal expression model
Mouse splenocytes were stimulated with Anti-Mouse CD3 (cat. no. 16-0031) and
Anti-Mouse CD28 (cat. no. 16-0281) Functional Grade Purified antibodies for 3 days,
with the addition of brefeldin A and monensin in the last 2 hours. The cells were stained
with Anti-Mouse CD8a PE-eFluor® 610 (cat. no. 61-0081), Anti-Mouse Ki-67 eFluor® 450
(cat. no. 48-5698), and Anti-Mouse Granzyme B PE-Cyanine7 (cat. no. 25-8898),
followed by hybridization of Type 4 Mouse Ki-67 Alexa Fluor ® 488 probe set
(cat. no. VB4-16518) and Type 6 Mouse Granzyme B Alexa Fluor® 750 probe set
(cat. no. VB6-16522). Cells in the lymphocyte gate or the mRNA+ events were
used for analysis.
30.6±7.6%
positive
18.6±7.1%
positive
Ki-67 mRNA
Alexa Fluor® 488
Granzyme B mRNA
Alexa Fluor® 750
Figure 8B
4500
4000
CV=15.0%
Tech 1
Tech 2
MFI of mRNA+ events
3500
Tech 3
3000
Tech 4
CV=12.6%
2500
2000
1500
1000
500
0
Ki-67
Granzyme B
Day-to-day variability
To understand day-to-day variation, human PBMC stimulated with the Cell Stimulation Cocktail (plus protein transport inhibitor
cocktail) were analyzed fresh, or cryopreserved and analyzed one week later. As shown below, the percentage of positive events is
virtually unchanged between the fresh and cryopreserved samples.
Day-to-day variation of
PrimeFlow™ RNA Assaya
IFNγ mRNAb
IFNγ proteinb
Experiment 1
58.0
90.3
Experiment 2
57.8
87.7
Average
57.9
89.0
Standard Deviation
0.14
1.84
CV (%)
0.24
2.07
a
Normal human PBMC were stimulated and analyzed immediately (Experiment 1) or were
cryopreserved and analyzed one week later (Experiment 2) for the expression of IFNγ
mRNA and protein. Samples were surface stained with Anti-Human CD8a PE-eFluor® 610
(cat. no. 61-0088) and followed by hybridization of human IFNγ Alexa Fluor® 750
(cat. no. VA6-13121).
b
6
Data represent the percentage of IFNγ+ events, gated on viable CD8+ cells.
Sensitivity and specificity
The central dogma of molecular biology states that DNA gives rise to RNA, which in turn gives rise to protein. However, studies have
shown that the correlation between levels of RNA and protein products vary widely. Here we demonstrate how PrimeFlow™ RNA
Assay can reveal the unique kinetics of mRNA and protein in the same cells to understand the correlation between the two over time,
and in response to stimulation. Human PBMC were stimulated with the Cell Stimulation Cocktail (plus protein transport inhibitors) for
as long as five hours. Using PrimeFlow RNA Assay, the cells were assessed for IFNγ or TNFα mRNA expression and protein in CD8+
or CD8- lymphocytes at hourly intervals. As shown below, both CD8+ and CD8- lymphocytes respond to stimulation, but IFNγ and
TNFα mRNA and protein each exhibit unique kinetics depending on the lymphocyte subset being analyzed. Of note, while IFNγ mRNA
is rapidly upregulated after one hour of stimulation, IFNγ protein is not detected until the second hour. In contrast, TNFα mRNA and
protein are both upregulated within the first hour, and although CD8+ cells maintain TNFα mRNA, the CD8- lymphocytes more rapidly
downregulate TNFα mRNA, and show a subsequent slow decline in TNFα protein levels. Thus, PrimeFlow RNA Assay enables the study
of gene expression at the single-cell level in heterogeneous samples without the need for sorting specific subsets and has the ability to
elucidate the kinetics of mRNA and protein expression.
IFNγ Protein eFluor® 450
IFNγ Protein eFluor® 450
protein
CD8+CD8+
protein
mRNA
CD8+CD8+
mRNA
CD8- protein
CD8- protein
CD8- mRNA
CD8- mRNA
IFNγ Protein eFluor® 450
Figure 9B
TNFα mRNA Alexa Fluor® 488
TNFα mRNA Alexa Fluor® 488
5 hr
TNFα Protein PE-Cyanine7
TNFα Protein PE-Cyanine7
5 hr
IFNγ Protein eFluor® 450
TNFα mRNA and protein expression in T cell subsets by FlowRNA
100
TNFα mRNA and protein expression in T cell subsets by FlowRNA
100
75
75
50
50
25
25
0
3 IFNγ
Hours post-smulaon
®
0
1
IFNγ0 Protein
eFluor
4502
Figure 9C
100
TNFα mRNA Alexa Fluor® 488
IFNγ Protein eFluor® 450
0
TNFα mRNA and protein expression in T cell subsets by FlowRNA
TNFα mRNA Alexa Fluor® 488
4 hr
IFNγ mRNA Alexa Fluor® 750
IFNγ Protein eFluor® 450
TNFα Protein PE-Cyanine7
Percent posive cells
3 hr
IFNγ mRNA Alexa Fluor® 750
TNFα Protein PE-Cyanine7
TNFα Protein PE-Cyanine7
Percent posive cells
TNFα mRNA Alexa Fluor® 488
TNFα mRNA Alexa Fluor® 488
TNFα Protein PE-Cyanine7
4 hr
IFNγ mRNA Alexa Fluor® 750
IFNγ Protein eFluor® 450
IFNγ Protein eFluor® 450
3 hr
IFNγ mRNA Alexa Fluor® 750
IFNγ Protein eFluor® 450
2 hr
IFNγ mRNA Alexa Fluor® 750
IFNγ Protein eFluor® 450
TNFα Protein PE-Cyanine7
IFNγ mRNA Alexa Fluor® 750
IFNγ mRNA Alexa Fluor® 750
1 hr
TNFα mRNA Alexa Fluor® 488
TNFα Protein PE-Cyanine7
TNFα Protein PE-Cyanine7
IFNγ mRNA Alexa Fluor® 750
TNFα Protein PE-Cyanine7
IFNγ mRNA Alexa Fluor® 750
IFNγ mRNA Alexa Fluor® 750
IFNγ mRNA Alexa Fluor® 750
CD8-
TNFα Protein PE-Cyanine7
2 hr
IFNγ mRNA Alexa Fluor® 750
TNFα mRNA Alexa Fluor® 488
TNFα Protein PE-Cyanine7
0 hr
CD8+
1 hr
TNFα mRNA Alexa Fluor® 488
CD8-
TNFα mRNA Alexa Fluor® 488
CD8+
TNFα mRNA Alexa Fluor® 488
0 hr
TNFα mRNA Alexa Fluor® 488
Figure 9A
1
4
5 ® 450
Protein
eFluor
2
3
Hours post-smulaon
4
5
IFNγ mRNA and protein expression in T cell subsets by FlowRNA
7525
500
0
1
2
3
Hours post-smulaon
4
CD8+ protein
CD8+ mRNA
protein
CD8+CD8protein
CD8- mRNA
CD8+ mRNA
CD8- protein
CD8- mRNA
5
25
Percent posive cells
50
IFNγ mRNA and protein expression in T cell subsets by FlowRNA
75
100
Percent posive cells
CD8+ mRNA
CD8- protein
CD8- mRNA
100
Percent posive cells
CD8+ protein
CD8+ mRNA
CD8- protein
mRNA
CD8+CD8protein
Percent posive cells
100
75
TNFα mRNA and protein expression in T cell subsets by FlowRNA
50
75
25
50
0
0
1
2
3
Hours post-smulaon
25
4
5
IFNγ mRNA and protein expression in T cell subsets by FlowRNA
100
0
0
0
1
2
3
Hours post-smulaon
4
5
0
1
2
3
4
5
CD8- protein
CD8- mRNA
Percent posive cells
Percent posive cells
CD8+ protein
75
Hours post-smulaon
CD8+ mRNA
50
CD8- protein
IFNγ mRNA and protein expression in T cell subsets by FlowRNA
Correlation andCD8kinetics
TNFα transcription and translation
mRNAof IFNγ and
10025
Normal human PBMC were stimulated
with Cell Stimulation Cocktail (plus protein transport inhibitors) (cat. no. 00-4975) for 0-5 hrs, then subjected to PrimeFlow™ RNA Assay.
Cells were intracellularly stained with Anti-Human
CD8α PE-eFluor® 610 (cat. no. 61-0088), Anti-Human IFNγ eFluor® 450 (cat. no. 48-7319), and Anti-Human TNFα PE-Cyanine7
0
CD8+ protein
75 0
1 Human
2 TNFα
3 Alexa4 Fluor ®5 488 probe set (cat. no. VA4-10289) (Figure 9B) and Human IFNγ Alexa Fluor ® 750 probe set
(cat. no. 25-7439),
followed by hybridization
with
Hours post-smulaon
(cat. no. VA6-13121)
(Figure 9C). CD8+ or CD8- cells in the lymphocyte gate were used for analysis.
CD8+ mRNA
50
25
7
Orthogonal validation
PrimeFlow™ RNA Assay was assessed by examination of genes previously measured by QuantiGene® Plex Assay, a hybridization-based
assay using the xMAP® Luminex® magnetic bead platform. As shown in Figure 10A, GAPDH showed the highest MFI in U937 cells,
consistent with the highest relative signal captured in QuantiGene Plex Assay. HMBS had the lowest MFI, consistent with gene expression
at low levels in U937 cells. Signal-to-noise ratios were calculated relative to the No Probe control sample and are shown in Figure 9B.
Next, we assessed the expression of the same genes in normal human lymphocytes and monocytes (Figure 9C). Although U937 is a
monocytic cell line, there were notable differences between primary monocytes and U937 cells. HMBS was completely undetectable in
primary human monocytes, and instead of GAPDH, B2M was the most highly expressed gene in monocytes. Primary human lymphocytes
diverged even further with very low levels of GAPDH and high levels of B2M. These data highlight the importance of understanding gene
expression levels in the cells of interest, as expression can vary even in cell lines derived from the same primary cell type. Furthermore, based
on QuantiGene Plex data, U937 cells are known to express 5-10 copies of HMBS mRNA, suggesting that in a fully optimized system it is
possible to detect 5-10 RNA copies by PrimeFlow RNA Assay.
HMBS
B2M
PPIB PPIB
B2M
PPIB
GAPDHGAPDH
Count
Count
Count
Count
GAPDH
No Probe
HMBS
B2M
PPIB
GAPDH
1400
140.00
AlexaAlexa
FluorFluor
647 647
AlexaFluor
Fluor ®
647
Alexa
647
Figure 10C
Lymphocytes
Lymphocytes
Lymphocytes
Monocytes
Monocytes
Monocytes
Monocytes
1000
100.00
800
80.00
68.9
56.1
60.00
600
400
40.00
200
20.00
1.0
3.6
0
No Probe
dapB
HMBS
B2M
PPIB
GAPDH
Count
Count
Count
Count
Count
Count
Count
Count
Lymphocytes
122.4
1200
120.00
Signal to Noise Ratio
HMBS HMBS
No probe
No probe
B2M
No probe
Figure 10B
U937U937
U937
U937
Signal-to-noise ratio
Figure 10A
0
NoProbe
probe No
HMBS
HMBS
B2M
B2M
PPIBPPIB
GAPDHGAPDH
Orthogonal validation of QuantiGene® Plex and PrimeFlowTM RNA Assays
U937 (Figures 9A and 9B) or normal human PBMC (Figure 9C) were hybridized to a
series of Type 1 Positive Control Alexa Fluor® 647 probe sets, following PrimeFlow™ RNA
Assay protocol. The signal-to-noise ratio was calculated as the ratio between the
MFI of the Positive Control gene and the MFI of the No Probe control.
AlexaAlexa
Fluor
647®647
Fluor
Alexa
Fluor
647
Alexa
Fluor®
647
Alexa
Fluor
647
Alexa
Fluor
647
Alexa Fluor 647
Alexa Fluor 647
Correlation of QuantiGene® Plex and TaqMan® data
To demonstrate the accuracy of QuantiGene® Plex assay, measurements of twenty transcripts from two reference RNA samples were
made and compared to QuantiGene® and TaqMan® data. Reference RNA samples include human brain total RNA and universal human
reference RNA as described in Canales et al. 2006. Nature Biotechnology 24(9):1115-1122. As shown below (Figure 11), excellent
correlation is noted in RNA level fold changes in the reference samples, between QuantiGene® Plex and TaqMan® assays (R2 = 0.965).
TaqMan® data are described in Canales, et al. 2006 publication. These studies show that TaqMan® based PCR, QuantiGene® Plex, and
PrimeFlow™ RNA Assay provide consistent results.
R2=0.965
Figure 11: Comparison of QuantiGene® and TaqMan® RNA level fold
changes in total and reference RNA.
4
3
TaqMan®
2
1
-4
-2
-1
2
-2
-3
-4
-5
QuantiGene® Plex Assay
8
4
Technology specificity
The bDNA technology achieves high target specificity with the use of oligonucleotide pairs—a design resulting in signal amplification
only when two adjacent target probe oligonucleotides (left oligonucleotide and right oligonucleotide) bind to the specific target. To
determine the assay specificity, probe sets for human GAPDH containing left oligonucleotides alone, or right oligonucleotides alone,
were tested and compared to a complete probe set in human U937 cells. Fluorescent signal is detected only with the complete probe
set, correlating to the abundant expression of GAPDH in U937 cells. In contrast, no signal is detected when either the left or right
oligonucleotides are used individually, similar to the No Probe control. Additionally, a negative control probe set for DapB, a bacterial
gene in Bacilus subtillis, yielded no specific signal.
Figure 12
Figure 13
U937
Label Probe
Label
Probe
Pre-amplifier
Pre-Amplifier
Count
Count
Amplifier
Amplifier
800
GAPDH Left + Right
GAPDH Left Oligos Only
GAPDH Right Oligos Only
dapB
No Probe
600
400
200
RNA
RNA
Left Oligo
Left Oligo
0
100
RightOligo
Oligo
Right
101
102
103
Fluorescent Signal
Fluorescent
signal
104
Figure 14
1200
1200
MFI
MFI
1000
1000
800
800
600
600
400
400
200
200
0
0
NoProbe
probe
No
Oligos Only
Oligos Only
GAPDH Left
GAPDHRight
Right
GAPDHLeft
Left
GAPDH
Left GAPDH
GAPDH
Oligos Only
Oligos Only
+ Right Oligos
Oligos Only Oligos Only +Right Oligos
Dapβ
DapB
Demonstration of specificity of the PrimeFlow™ RNA Assay
Illustration of a Probe Set design containing both left and right oligonucleotide pairs, and signal amplifications reagents, Pre-amplifier, Amplifier, and Lable Probes
(Figure 12). Flow cytometry histogram data of GAPDH RNA expression in U937 cells (Figure 13). Quantitative representation of GAPDH signal with different probe sets
based on Mean Fluorescent Intensity (MFI) (Figure 14).
9
Assay channel sensitivity
PrimeFlow™ RNA Assay is capable of detecting three different RNA targets in a single cell through the use of three different
fluorochromes. However, each fluorochrome provides different levels of sensitivity. To understand the relative sensitivity of the three
RNA detection channels, we assessed the expression level of the same gene in each of the three probe set types using U937 samples
or in mouse splenocytes. Human B2M probe sets in Type 1 (Alexa Fluor® 647), Type 4 (Alexa Fluor® 488), or Type 6 (Alexa Fluor® 750)
were used to detect B2M RNA targets in human U937 cells (Figure 15A). The signal-to-noise ratio relative to the No Probe control
was calculated and the relative sensitivity to Type 6 (Alexa Fluor 750) probe set was assessed. As shown in Figures 15A and 15B,
Type 1 (Alexa Fluor 647) provides the greatest sensitivity. We observed similar sensitivity when assessing the expression of β-Actin
in mouse splenocytes. For this reason, Type 1 probes for genes with low or unknown levels of expression. Type 4 (Alexa Fluor 488) and
Type 6 (Alexa Fluor 750) probe sets have similar sensitivity and are recommended for genes with medium to high levels of expression.
As with any multi color flow cytometry experiment, actual results depend on instrument configuration, detector sensitivity,
instrument settings, and compensation, all of which require optimization to obtain the best results.
Human B2M mRNA relative sensitivity (%)
Figure 15A
600%
600%
Assessing assay channel sensitivity
U937 cells were hybridized with B2M probe sets in each probe set type to assess
the different sensitivity of each channel. Values above each bar represent the
signal-to-noise ratio. Total viable cells were used for analysis (Figure 15A).
Mouse splenocytes were hybridized with β-Actin (ACTB) probes, in triplicate.
Total viable cells were used for analysis (Figure 15B).
58.1
500%
500%
400%
400%
300%
300%
200%
200%
13.4
100%
100%
10.7
0%0%
Type 1
AF647
Type 4
AF488
Type 6
AF750
AF=Alexa Fluor®
Figure 15B
Alexa FluorAlexa
647 Fluor
647Fluor 647
Alexa
ACTB mRNA
10
Alexa FluorAlexa
647 Fluor
647Fluor 647
Alexa
Count
Count
Count
Alexa Fluor® 750
Count
Count
Count
Count
Alexa Fluor® 488
Count
Count
Count
Count
Count
Alexa Fluor® 647
Alexa FluorAlexa
647 Fluor
647Fluor 647
Alexa
Sample types tested
PrimeFlow™ RNA Assay has been validated for use with suspension cells such as peripheral blood mononuclear cells (cryopreserved,
stimulated cells and freshly isolated cells), mouse bone marrow cells, mouse tissue (fresh, stimulated and cryopreserved), cultured
mammalian leukemic cell lines, and adherent cells.
Cell
Recommended Positive Control Gene
Human lymphocytes (PBMC) – fresh and cryopreserved
RPL13a, B2M
Human monocytes (PBMC) – fresh and cryopreserved
RPL13a, B2M
Mouse splenocytes (tissue) – fresh and cryopreserved
ACTB, RPL13a
Mouse thymocytes (tissue)
ACTB, RPL13a
Mouse bone marrow
ACTB
Human monocytic lymphoma, U937
RPL13a, B2M
Human T cell lymphoma, Jurkat
RPL13a, B2M
Human cervical carcinoma, HeLa*
RPL13a, GAPDH
Human lung carcinoma, PC9*
RPL13a, GAPDH
*adherent cells
Adherent cell data
Figure 16: Human cervical carcinoma cells, HeLa vs Human lung
adenocarcinoma cells, PC9
Adherent cell lines, HeLa and PC9, were detached using either EDTA or typrsin
and then subjected to PrimeFlow™ RNA Assay. HeLa and PC9 cells were hybridized
with human KRT19, PPIB, and GAPDH probe sets.
Helical human cervical carcinoma cells, HeLa vs human lung adenocarcinoma cells, PC9
*adherent cells
120
120.00
HeLa-Trypsin
HeLa-EDTA
PC9-Trypsin
PC9-EDTA
Signal-to-noise ratio
100
100.00
80
80.00
60
60.00
40
40.00
20
20.00
0
0.00
KRT19
KRT19
PPIB
PPIB
GADPH
GAPDH
Helical human cervical carcinoma cells, HeLa vs human lung adenocarcinoma cells, PC9
11
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