To understand systems-level biological responses, a large RNA expression levels traditionally have been measured
number of transcriptional measurements from diseased tis-sues, and from tissues and cells treated with pharmaceuticalagents, have been made using numerous genome-wideexpression proWling technologies [1–3]. To develop theseobservations into combinatorial biomarkers for drug devel-opment and clinical assessment of disease susceptibility andprogression, simple, robust, and Xexible proWling technol-ogy is needed to ensure more accurate characterization ofgene expression changes from a broad population of bio-logical specimens while retaining a capability for high-throughput analysis.
using Northern blot and nuclease protection assays. How-ever, these approaches have limited sensitivity and the datagenerated are more qualitative than quantitative in nature.Greater sensitivity and quantiWcation is possible withreverse transcription polymerase chain reaction (RT-PCR)2-based methods, such as quantitative real-time
Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60 51
RT-PCR, but these approaches have low multiplex capabil-ities and are especially sensitive to DNA contaminationand ampliWcation eYciency diVerences between samples[4,5]. Microarray technologies, which have revolutionizedgenomics and spawned the Weld of systems biology, arewidely used in discovery research. However, the relativelylong experimental procedure, sensitivity in measuringdiVerential expression limited to large fold changes, suscep-tibility to variations in both intrinsic (e.g., reagent andenzyme quality, purity of initial RNA) and extrinsic (e.g.,microarray systems, controls, experimental design, person-nel training) experimental factors, as well as the computa-tional complexity of screening for false positives havelimited its use in high-throughput expression proWling andclinical applications [6].
Most current methods used to detect and quantitatemessenger RNA (mRNA) expression levels from experi-mental samples require RNA isolation, reverse transcrip-tion, and in most cases target ampliWcation. Eachprocedural step following sample procurement has thepotential to introduce variability that leads to low overallassay precision [4]; therefore, it is desirable to minimize theprocessing steps necessary to obtain quality measurements.Recently, two multiplex screening assays that quantitatemRNA directly from crude cell lysates were reported. TheWrst combines nuclease protection with luminescent arraydetection [7]. Although no RNA puriWcation is required,this assay showed relatively low assay reproducibility anddetection sensitivity. The second multiplex RNA quantiW-cation assay uses electrophoretic tags in the InvadermRNA assay [8]. This technology requires enzymatic reac-tion for RNA quantiWcation, which could aVect the assayreproducibility due to potential inconsistency of theenzyme performance in the context of diVerent cell lysates.
New technologies are required to bridge the gap amongassay sensitivity, throughput, and ease of use while settingthe highest standards for data quality. The branched DNA(bDNA) assay is a sandwich nucleic acid hybridizationassay that provides a unique approach for mRNA quantiW-cation by amplifying the reporter signal rather than targetsequences [9]. By measuring mRNA directly from crude celllysates and tissue homogenates, this assay avoids errorsintroduced during the extraction and ampliWcation of tar-get sequences. The bDNA-based VERSANT assay is Foodand Drug Administration approved for clinical manage-ment of HBV, HCV, and HIV viral load determination [10–12], and it has been adapted for discovery and developmentresearch of drug metabolism [13], structure–activity rela-tionships [14], high-throughput screening [15], and smallinterfering RNA (siRNA) knockdown analysis [16]. Simi-larly, the Luminex bead array system has been used in awide range of multianalyte applications throughout thedrug discovery and diagnostics Welds and is widely adoptedfor quantitative multiplex protein expression analysis [17–19]. At the core of the xMAP technology are 100 discretebeads, each with a unique Xuorescent signature, that arecoupled with a capture reagent speciWc to a particular
bioassay, enabling the detection of up to 100 diVerentanalytes from a single sample.
We report here the development of a multiplex bDNAassay that combines the robust expression measurementcapabilities of the bDNA assay with the multiplex capabil-ity of the Luminex platform by coupling xMAP Xuorescentbeads with a set of oligonucleotide capture probes. In sodoing, we discovered that exceptionally high assay speciWc-ity can be obtained by taking advantage of cooperativehybridization, a strong and stable hybridization formed bythe collective force of multiple weak and unstable hybrid-ization interactions, between capture probes and gene-spe-ciWc probe sets. This new mRNA quantiWcation methodmeasures the expression levels of multiple mRNA tran-scripts quantitatively and allows discrimination betweengenes that are more than 95% homologous from puriWedRNA and crude cell lysates with high accuracy and repro-ducibility. The simplicity and data quality of the assaymake it an ideal tool for high-throughput parallel quantita-tive gene expression analysis.
Materials and methods
In vitro transcribed RNA
Complementary DNA clones encoding the full-lengthtarget genes were obtained commercially and used as tem-plates for in vitro transcription (IVT) to generate RNAstandards (Invitrogen, Carlsbad, CA, USA; Origene, Rock-ville, MD, USA; and Open Biosystems, Huntsville, AL,USA). IVT RNA was transcribed from 1.5 �g of linearizedplasmid via T3, T7, or SP6 promoters using the Ampli-scribe kit (Epicenter, Madison, WI, USA) and was quanti-Wed using RiboGreen Xuorescence (Molecular Probes,Eugene, OR, USA) and the Envision 2100 multilabel reader(PerkinElmer, Wellesley, MA, USA). IVT RNA transcriptsfor each panel were mixed and serially diluted fourfold togenerate a standard curve with target RNA levels rangingfrom 2.4£ 104 to 9.6£107 transcripts (0.04–160 amol). TheIVT RNAs were used as reference standards to assess assaysensitivity, speciWcity, accuracy, and linear dynamic range.
Probe set design for single-plex and multiplex bDNA assays
All bDNA assays use three sets of pooled oligonucleo-tides that function to capture, provide binding sites forsignal ampliWcation molecules, and stabilize the mRNAof interest. The capture extender (CE) pool contains oli-gonucleotides that are complementary to the mRNA ofinterest and to capture probes (CPs) covalently linked to asolid support (either plate or microsphere [see below]).The label extender (LE) pool has oligonucleotides thatcontain sequences complementary to the mRNA of inter-est and to the bDNA ampliWer molecule. The blocker(BL) pool contains oligonucleotides that are complemen-tary to the mRNA where neither CE nor LE bind andprotects the mRNA by maintaining an intact RNA–DNA
52 Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60
hybrid. Probe design software [20] was modiWed to gener-ate probe sets for use in single-plex or multiplex bDNAassays. For each target sequence, the software algorithmidentiWes regions that can serve as annealing templates forCEs (5–7/gene), LEs (10–15/gene), or BLs. NonspeciWchybridizations between CE and LE, between CE andbDNA, between CE and label probe, and between LE andCP were minimized by removing probes when interactionshad a high negative �G (<¡7.0 kcal/mol). Several multi-plex panels were developed by mixing the CE, LE, and BLfor each of the desired target genes (Table 1). Except forthe portion of the CE probe that hybridizes with CP,probe set designs are essentially the same for both single-plex and multiplex assays.
Single-plex bDNA assay
Single-plex bDNA assays were performed according tothe QuantiGene Reagent System instruction manual(Genospectra, Fremont, CA, USA). BrieXy, cultured cellsand media were harvested by adding lysis mixture (33%Wnal concentration) and were incubated for 15 min at37 °C, followed by the addition of target gene probe setmix (CE, 0.165 fmol/�l; LE, 0.66 fmol/�l; BL, 0.33 fmol/�l).Cell lysates (100 �l) were transferred to a CP-coated welland then incubated for 16 h at 53 °C. For measuring IVTRNAs of known concentration (see below), a masterhybridization mixture was made prior to IVT RNA addi-tion. The components in a 100-�l IVT RNA assay were33% lysis mixture, 40% capture buVer (Genospectra),100 ng tRNA, and the gene-speciWc probe set. Signalsfrom bound mRNA–probe set complexes were developedby sequential hybridization with bDNA ampliWer andalkaline phosphatase-conjugated label probe at 46 °C for1 h each. Two washes with wash buVer (0.1£ SSC, 0.03%lithium lauryl sulfate) were used to remove unboundmaterials after each hybridization step. Dioxetane, analkaline phosphatase substrate, was added to wells andincubated at 46 °C for 30 min. Luminescence from eachwell was detected using an Lmax microtiter plate lumino-meter (Molecular Devices, Sunnyvale, CA, USA).
Cooperative hybridization
To determine the eVect of CP length on cooperativehybridization, three oligonucleotide CPs (CP16, 16-mer,5�-NH2-C6-ACTTTCTTTCCAAGAG-3�; CP15, 15-mer, 5�-NH2-C6-ACTTTCTTTCCAAGA-3�; and CP14, 14-mer, 5�-NH2-C6-ACTTTCTTTCCAAG-3�) complementary to theCE tails were covalently conjugated to capture plates, respec-tively, following a published procedure [21] and were used inbDNA assays measuring known quantities of interleukin 6(IL6) IVT RNA. To determine the eVects of CP length onnoncooperative hybridization, an oligonucleotide probe (CE-LE, 5�-AGGCATAGGACCCGTGTCtttttCTCTTGGAAAGAAAGT-3�) that combines sequences complementary toCP (CE tail) and to the bDNA molecule (LE tail) was used inthe bDNA assay in place of the probe set and target RNA.An oligonucleotide probe of the CE tail only (CE, 5�-CTCTTGGAAAGAAAGT-3�) that does not bind bDNA ampli-Wer was used as a negative hybridization control.
CPs for multiplex assay and coupling to solid supports
Unique 15-mer CP sequences were designed to minimizesecondary structure and cross-hybridization among eachother’s complementary sequences. Each was homologyscreened against current human, mouse, and rat databases(BLAST and NCBI) as well as against sequences locatedwithin the bDNA ampliWer and label probe molecules. Oligo-nucleotide CPs were synthesized with 5�-NH2-C6 linker (Bio-search, Novato, CA, USA) and covalently linked tocarboxylated Xuorescent-encoded microspheres (Luminex,Austin, TX, USA) [22]. BrieXy, 1.25£107 beads were resus-pended in 100�l of 0.1 M MES (pH 4.5) and were incubatedin the presence of approximately 8�M CP and 2 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(Pierce, Rockford, IL, USA). Crosslinking eYciency andhybridization speciWcity of each capture bead were evaluatedby hybridizing a single CP-complementary biotinylated oli-gonucleotide to a mix of 10 CP-coupled beads under thesame conditions as used in the multiplex bDNA assay forIVTs.
Table 1Target mRNAs analyzed using the three multiplex bDNA assay panels
Note. N/A, not applicable.
Bead number Cytokine panel Apoptosis panel 1 Apoptosis panel 2
Target symbol Accession number Target symbol Accession number Target symbol Accession number
Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60 53
Multiplex bDNA assay
Samples containing IVT RNA, total RNA, or celllysates were mixed with pooled multiplex probe sets andcapture beads (2000 beads/gene/assay) and hybridized for16 h at 53 °C in 100 �l volume. The components in a 100�lIVT RNA assay were 33% lysis mixture, 40% capturebuVer (Genospectra), 1 �g tRNA, and the panel-speciWcprobe set (CE, 0.165 fmol/�l/gene; LE, 0.66 fmol/�l/gene;BL, 0.33 fmol/�l/gene). Hybridization reactions weretransferred to a 0.45-�m Wlter plate (Millipore, Billerica,MA, USA), followed by sequential hybridization at 46 °Cwith bDNA ampliWer and 5�-dT(biotin)-conjugated labelprobe. Unbound materials were washed from beads (com-plexed with probe set and mRNA) through alternatingWltration and the addition of wash buVer (0.1£ SSC,0.03% lithium lauryl sulfate). Two washes were performedafter each hybridization step. After a Wnal wash, streptavi-din-conjugated R-phycoerythrin (SAPE) was added andincubated at room temperature for 30 min. The beadswere washed to remove unbound SAPE, followed by anal-ysis with a Luminex 100 IS system (Luminex) or a Bio-Plex system (Bio-Rad, Hercules, CA, USA).
Cell lysates for cytokine panel evaluation
U937 cells (American Type Culture Collection, Manas-sas, VA, USA) at an approximate density of 1 to 2 £ 105
cells/ml were allowed to diVerentiate in SFM media(Invitrogen) with 100 nM phorbol-12-myrstyl-13-acetate(PMA, Sigma, St. Louis, MO, USA) for 48 h. DiVerenti-ated cells, which become adherent, were stimulated withlipopolysaccharide (LPS, Sigma) at a concentration of1 �g/ml in RPMI growth media (Invitrogen) with 10%fetal bovine serum (FBS) for 4 h. After cell counting, thecells were directly lysed in the culture Xask by the additionof lysis mixture to the culture media and were incubatedat 37 °C for 15 min.
Cell lysates for apoptosis panel evaluation
HeLa cells (American Type Culture Collection) werecultured in Dulbecco’s modiWed Eagle’s medium (Invitro-gen) at an approximate density of 1 to 2£106 cells/ml andwere treated with recombinant human tumor necrosis fac-tor-� (TNF�, 1 ng/ml, R&D Systems, Minneapolis, MN,USA) or 1% bovine serum albumin/phosphate-buVeredsaline (BSA/PBS) vehicle control. Both TNF�-treated andvehicle-treated cells were harvested at 1, 3, and 6 h post-treatment by the addition of lysis mixture directly to theculture Xasks to generate cell lysates suitable for both sin-gle-plex and multiplex assays.
Data analysis and statistics
Three replicate assays (nD3) were performed for alldescribed experimental samples except where noted.
All multiplex data were derived from measuring medianreporter Xuorescence from 100 beads per gene per wellassayed and are represented as median Xuorescence inten-sity (MFI). Background signals were determined in theabsence of target mRNAs and were subtracted from signalsobtained in the presence of target mRNAs. The sensitivityof the assay for each target RNA was evaluated by deter-mining the limit of detection (LOD), deWned as the targetconcentration at which the signal is 3 standard deviationsabove the background. The correlation between the single-plex and multiplex bDNA assays was assessed throughmatched-pair analysis of same-sample data from single-plex and multiplex bDNA assays using JMP software (SASInstitute, Cary, NC, USA). Statistical signiWcance of bio-logical studies was tested using the Student’s t test or analy-sis of variance where appropriate (P < 0.01).
Results
Overview of the multiplex bDNA assay
To develop the multiplex bDNA assay, we used the Xuo-rescently encoded microspheres from Luminex as the solidsupport for the capture of speciWc mRNA species (Fig. 1).The ability to quantify multiple mRNA transcripts lies inthe design of a speciWc probe set for each mRNA transcript.A probe set consists of three types of synthetic oligonucleo-tide probes (CEs, LEs, and BLs) that hybridize to contigu-ous regions of the target mRNA molecule. CEs and LEsboth contain “tail” regions that are available for furtherhybridization. An intended target mRNA is captured to aspeciWc bead through the hybridization of multiple mRNA-bound CE tails to the complementary CP covalentlyattached to the bead. Signal ampliWcation occurs when themRNA-bound LE tails capture branched DNA moleculesthrough hybridization, and this in turn captures multiplebiotinylated label probes. Signals are generated by the bind-ing of SAPE to biotinylated label probes. Bead Xuorescentcolor codes and SAPE reporter signals from all beads in theWnal hybridization mixture are determined using the Lum-inex Xow cytometer, which maps each bead to a speciWcmRNA assay and provides a Xuorescence measurement ofSAPE reporter associated with that bead. The SAPE sig-nals are proportional to the number of mRNA transcriptscaptured to the beads.
Cooperative hybridization
When the assay temperature exceeds the melting temper-ature of two complementary nucleic acids, dissociation(rather than annealing) of the strands is favored (Fig. 2A,left). However, during the development of the multiplexbDNA assay, we discovered that a series of multiple unsta-ble hybridizations favor a stable mRNA capture throughcooperativity of hybridization (Fig. 2A, right). We have lev-eraged this “cooperative hybridization” to design probesets that drive highly stable and speciWc capture of a target
54 Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60
mRNA to a CP-conjugated solid support. In this system, anumber of CEs are designed to hybridize to several sitesalong the length of the target mRNA (Wve to seven CEs permRNA). To demonstrate cooperative hybridization, threeCPs varying in length from 14- to 16-mer DNA sequencewere designed and used for two experiments in a plate-
based, single-plex bDNA assay. CP14, CP15, and CP16contain 14 identical bases at their 5� ends, with CP15 andCP16 lengthened at the 3� ends by the addition of one andtwo bases, respectively. All probes have calculated meltingtemperatures below the 53 °C hybridization temperature.Noncooperative hybridization between a single CE and CP
Fig. 1. Overview of the multiplex bDNA assay. The three major steps of the multiplex bDNA assay are (i) capture of the speciWc mRNA transcripts totheir corresponding beads through CP–CE hybridization interactions, (ii) two serial hybridizations that capture the bDNA ampliWer and biotinylatedlabel probe molecules, and (iii) a hybridization that captures SAPE. The Luminex beads are then analyzed with a Luminex 100 IS system. The SAPE Xuo-rescence signal measured from each bead is proportional to the number of captured mRNA transcripts.
Target mRNA (cell lysate or tissuehomogenate)
Capture Beads
Label Extenders (LEs)
Blocking Probes (BLs)
Capture Extenders (CEs)
Amplifier(bDNA)
Label Probe(Biotinylated)
SAPE (Streptavidin-phycoerythrin)
Step 1 Step 2 Step 3
LEBL
mRNACE
CaptureProbe
CaptureBead
Fig. 2. Illustration that cooperative hybridization enables stable and speciWc capture of target mRNA. (A) In noncooperative hybridization conditions(left), when the hybridization temperature is above the calculated melting temperature of a CP–CE duplex, dissociation is favored and a stable capture ofthe target mRNA does not occur. However, the collective force of multiple hybridizations (right) under identical conditions favors the capture and stabil-ization of the target mRNA to the solid support. (B) Hybridization strength between CP14, CP15, CP16 with a CE–LE oligonucleotide is dependent onlength (melting temperature) of CPs under noncooperative conditions, as described in Materials and methods. CP-N is a nonspeciWc CP that is not com-plementary to CE–LE. CE is an oligonucleotide that binds to CP but not to bDNA ampliWer. (C) Cooperative hybridization permits stable capture of IL6IVT RNA at various concentrations independent of CP sequence length, as described in Materials and methods. Single-plex data are average relativeluminescent units (RLU), and the error bars represent the standard deviations of the average measurements.
Target mRNA
0.1
1
10
100
1000
10000
CP15CP14CP-NCapture Probe
RLU
CE-LECE
0.1
1
10
100
1000
IL6 IVT RNA (amol)
RLU
0.01 0.1 1 10 100
CP16CP15CP14
CP16
5'5' 3' 3'
53oC 53oC
CPCE
A
B C
Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60 55
was evaluated through an oligonucleotide (CE–LE) thatcontains the CE tail sequence complementary to the CPand the LE tail sequence complementary to a sequence inthe bDNA molecule (Fig. 2B). The strongest assay signalswere obtained with the longer 16-mer CP, whereas the 15-mer CP resulted in a 100-fold weaker signal, indicating aweaker hybridization interaction under these conditions.Minimal hybridization occurred with the 14-mer CP. Whenthese three CPs were used in a bDNA assay where multipleCEs are present in the probe set (Fig. 2C), overlappingassay signal was observed regardless of the CP length. Thissuggests that cooperative hybridization plays a prominentrole in capturing mRNA in the bDNA assay, especiallywhen the CP is the short 14-mer.
Coupling the novel principle of cooperative hybridizationwith the knowledge of sequence speciWcity allowed us todevelop a set of CPs that work together with little nonspeciWccross-hybridization. Each of 10 unique 15-mer CPs waschemically crosslinked to the surface of 10 diVerent, Xuores-cently encoded beads. Once coated with a speciWc CP, the CP-conjugated beads were pooled to make the bead array andwere tested for speciWc and cross-hybridization using biotinyl-ated oligonucleotides complementary to each CP (Fig. 3). Theassay signals of all possible nonspeciWc hybridizations wereless than 0.1% of those observed for the perfectly matchedpair, indicating a very high degree of hybridization speciWcitysuYcient for the capture of target mRNAs.
Performance evaluation of the multiplex bDNA assay
To demonstrate the performance of the multiplexbDNA assay, we developed several 10-plex panels (Table 1)
Fig. 3. Cross-reactivity of the CPs. The CPs were evaluated by hybridiza-tion with biotin-labeled complementary probes. Each hybridization con-tained a mixture of 10 CP-conjugated beads and 16.5 fmol of anindividual biotinylated complementary probe. Signals were generated byhybridization with SAPE as described in Materials and methods. Data arebackground-subtracted average MFI (net MFI).
CP1 CP2 CP5 CP6 CP14 CP20 CP21 CP22 CP24 CP25
CZ1
CZ2
CZ5CZ6CZ14CZ20CZ21CZ22CZ24CZ25
0
2000
4000
6000
8000
10000
12000
14000
Net
MF
I
Capture Probe No.
Bio
tinyl
ated
Com
plem
ent
and evaluated each for sensitivity, linear dynamic range,precision, and speciWcity using IVT RNA transcripts as ref-erence standards. The performance evaluation data aredetailed for the cytokine panel and summarized for theapoptosis panels.
To determine the cross-reactivity between targetmRNAs and probe sets within the full panel, 40 amol ofeach IVT RNA transcript was individually hybridizedwith the bead array and probe sets for all 10 genes in themultiplex bDNA assay. Cross-reactivity is expressed asthe percentage of the signal generated by hybridization ofmRNA to nontarget beads in the panel in relation to thespeciWc hybridization signal from the intended target. Thecross-reactivity between target mRNAs in the panel wasless than 0.1%, indicating minimal cross-hybridizationbetween the genes in the panel (Fig. 4A and Table 2). Theassay speciWcity was evaluated by measuring the signals ofBacillus subtilis dapB IVT RNA (little homology withhuman RNA) and human IL10 IVT RNA (undetectableexpression in human U937 cells) in a complex mixturethat included U937 total RNA (Fig. 4B). The IVT RNAtranscripts of dapB and IL10 were serially diluted four-fold from 160 to 0.04 amol and mixed with 0.2 �g of totalRNA extracted from human U937 cells prior to perform-ing the multiplex bDNA assay. Overlapping signals wereobserved through the entire dilution series for both dapBand IL10, suggesting that minimal nonspeciWc hybridiza-tion occurred in the presence of U937 total RNA. Impor-tantly, the addition of 0.2 �g of total RNA did notincrease the assay background, further demonstrating theassay speciWcity. Tests that determine spike recovery of atarget from a complex mixture is a typically recom-mended analytical procedure to assess assay accuracy.The assay is considered reliable if the signal diVerencebetween pure and spiked-in analyte is within §20%. Inthis case, no change in assay signal was observed in thepresence of U937 total RNA, indicating that nonspeciWcRNA does not seem to interfere with the hybridization ofthe target RNA to the capture beads. As a result, IVTRNA standard curves can be used to quantify the abso-lute copies of RNA transcripts in a sample.
To determine the assay sensitivity and linear dynamicrange, 10 target IVT RNA transcripts were equally mixedand serially diluted fourfold to generate a standard curvewith target RNA levels ranging from 2.4£ 104 to 9.6£ 107
transcripts (0.04–160 amol). The signal responses for all 10targets were linear across the target concentration rangeexamined with coeYcients of correlation (R2) averaging0.99, indicating that the linear dynamic range of the assayspans more than 3 logs (Fig. 4C). The sensitivity of theassay was evaluated for each target RNA by determiningthe LOD, deWned as the target concentration at which thesignal is 3 standard deviations above the background. Thecalculated LOD was 0.06 amol for vascular endothelialgrowth factor (VEGF) and 0.04 amol or lower for theremaining cytokines and glyceraldehyde phosphodehy-drogenase (GAPD) (Table 2).
56 Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60
d Assay performed using Bio-Plex system calibrated with high sensitivity se
ttings.
Fig. 4. Performance evaluation of the cytokine panel in a multiplex bDNA assay. (A) Cross-reactivity was evaluated by hybridizing individual IVT RNAtranscripts (40 amol, represented by gene name on x axis) with a mixture of 11 CP-conjugated beads. Data are background-subtracted average MFI (netMFI). (B) SpeciWcity was evaluated by hybridizing increasing amounts of dapB or IL10 IVT in the presence or absence of 0.2 �g of total RNA. (C) Sensi-tivity and dynamic range were evaluated using serially diluted IVT RNAs for all genes in the panel. The average coeYcients of correlation (R2) of the sig-nals exhibited a linear response with respect to dilution (R2 D 0.99).
IL10 IVT RNA (amol)
Net
MF
I
0.01 0.1 1 10 100 10001
10
100
1000
10000
100000
IVTIVT + Total RNA
dapB IVT RNA (amol)
Net
MF
I
0.01 0.1 1 10 100 10001
10
100
1000
10000
100000
IVTIVT + Total RNA
IL8
VEGF
IFNG
IL2 IL6
GAPD
CSF2IL1
0
TNFAIL1
B
dapB
1819
2022
2526
2728
2933
34
0
5000
10000
15000
20000
25000
30000
Net
MF
I
IVT RNA
Bea
d N
umbe
r
1
10
100
1000
10000
100000
0.01 0.1 1 10 100 1000
CSF2dapBGAPDIFNGIL10IL1BIL2IL6IL8TNFAVEGF
Net
MF
I
IVT RNA (amol)
B
A
C
Table 2Detection sensitivity, cross-reactivity, assay background, and assay precision of the three multiplex bDNA assay panels
a Limit of detection (LOD) of a target is the concentration at which the signal exceeds 3 standard deviations above the background signal.b Cross-reactivity is the percentage of signal generated by hybridization of mRNA to nontarget beads, in relation to the speciWc hybridization to its
target bead (100%).c Assay performed using Luminex 100 IS system.
Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60 57
Assay precision between diVerent wells (intraplate) andbetween assays performed on diVerent days (interplate) wasassessed by calculating coeYcients of variation (CVs) foreach gene expression measurement from 0.04 to 160 amolIVT RNA across multiple samples. Precision values foreach gene were measured across multiple wells within a sin-gle plate (nD 4) and across multiple plates (nD 3). Theintraplate CV average was 8% and ranged from 5 to 14%,whereas the interplate CV average was 14% and rangedfrom 7 to 22% (Table 2). An average intraplate CV of lessthan 10% has been routinely obtained using triplicate sam-ples. It should be noted that the CVs were highly compara-ble from high to low concentrations of the target IVT RNAtested, suggesting that accurate quantiWcation can beachieved in a target concentration-independent fashion.
Regulation of gene expression from cellular models of inXammation and apoptosis
We demonstrated the utility of this assay using celllysates from two well-characterized cell culture model sys-tems that elicit expression changes in genes within our pan-els. The Wrst model system used PMA/LPS to inducechanges in cytokine gene expression of inXammatoryresponses in U937 cells [23], and the second model systemused TNF�-treated HeLa cells to monitor expressionchanges in pro- and anti-apoptotic genes [24].
U937 cells with or without PMA/LPS treatment werelysed in the culture Xasks by adding a lysis buVer (Geno-spectra QuantiGene kit) directly to the culture media. Thelysis buVer facilitates the release and hybridization ofintact mRNA, and crude cell lysates were used directly forall multiplex bDNA assays. In untreated U937 cells,GAPD and VEGF were expressed at high levels; IL8,IL1� (IL1B), and TNF� (TNFA) were expressed at mod-erate to low levels; and IL6, IL10, CSF2 (GM–CSF), IL2,and IFN-� (IFNG) were below the assay detection limit(Fig. 5A). PMA/LPS treatment strongly induced theexpression of proinXammatory cytokines IL8, IL1�, andTNF�. However, cytokines IL6, IL10, and CSF2 wereonly moderately induced, whereas IFN-�, IL2, and VEGFremained essentially unchanged. The average CVs of themeasurements (including both signal and background)were 12.8 and 8.9% for uninduced cells and induced cells,respectively. Further veriWcation of mRNA levels fromthe same cell lysates was performed using the well-estab-lished single-plex bDNA assay. The results showed thesame pattern of gene expression with a correlation factorof 0.94 by matched pair analysis (Fig. 5B). In the multi-plex assay, the signals of the induced genes increased line-arly with the increasing amount of cell lysates (6000–25,000 cells) assayed, whereas the background levelsremained the same throughout the assay, demonstratingthe speciWc nature of the multiplex assay signal (Fig. 6).These results are in agreement with the previouslyreported U937 cell expression proWle following inductionby PMA/LPS treatment [23].
To further demonstrate the utility of the multiplex assay,we developed two 10-plex apoptosis panels and analyzedthe expression of these genes from HeLa cells treated withTNF� for 1, 3, or 6 h. Treatment with TNF� induced therapid translocation of NF�B complex from cytoplasm tothe nucleus within 30 min, where it stimulated the expres-sion of downstream apoptosis mediators [24]. The Wrst
Fig. 5. Induction of cytokine gene expression following PMA/LPS treat-ment of U937 cells. (A) Gene expression from control and PMA/LPS-treated cells was measured from 40,000 cell-equivalent lysates using the10-plex cytokine panel. When the signal for a gene was below the detec-tion limit, the expression level of that gene was not plotted. (B) Geneexpression was measured, using the 20,000 cells equivalent from the samecell lysates, in a single-plex bDNA assay. The measurements were per-formed in quadruplicate, and data were normalized to 40,000 cells equiva-lent. In both panels A and B, the control and PMA/LPS-treated samplesare normalized to GAPD. RLU, relative luminescent units. Error barsrepresent the standard deviations of the average responses.
110
1001000
10000100000 Multiplex
IL1B
Net
MF
I
IL8
GAPDCSF2
VEGFIL
2TNFA
IL6
IL10
IFNG
ControlPMA/LPS
110
1001000
10000100000 Single-plex
IL1B
Net
RLU
IL8
GAPDCSF2
VEGFIL
2TNFA
IL6
IL10
IFNG
ControlPMA/LPS
A
B
Fig. 6. Linear response of assay signals scale with sample input. Geneexpression from serially diluted PMA/LPS-induced and control celllysates was measured using the 10-plex cytokine panel. The assay signalincreased linearly as the number of induced cells in the lysate increased.Expression from the uninduced cells remained unchanged as cell numbersin the lysate increased. Error bars represent the standard deviations of theaverage measurements.
0
20
40
60
100
U937 Cells (x 103)
Net
MF
I
0 5 15 25 3010 20
80
CSF2 Induced
IL10 Induced
CSF2 Uninduced
IL10 Uninduced
58 Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60
apoptosis panel measures the expression of several majorsubunits of the NF�B transcription complexes, includingRELA, RELB, NFKB1, NFKB2, NFKB1A, a number ofadditional pro- and anti-apoptotic regulators, and a num-ber of cytokines (Table 1). Several patterns of gene expres-sion were observed (Fig. 7A). The expression of TNFAIP3(A20) and TNF� was rapidly elevated after 1 h TNF� stim-ulation and then declined between 3 and 6 h. The expressionof NFKB1, NFKB2, NFKB1A, and IL6 increased after 1 hTNF� stimulation and maintained roughly the same levelof expression throughout the course of the experiment. Theexpression of RELB increased moderately at 1 h; however,it elevated to much higher levels after 3 and 6 h TNF� stim-ulation, consistent with the inhibitory role it plays in limit-ing the NF�B transcriptional response. Also consistentwith their known functional role in the NF�B transcriptioncomplex and in cell cycle control, the expression levels ofRELA and CDKN1A did not change signiWcantly onTNF� treatment [24]. The average percentage CV of allexpression measurements was 8.5%. VeriWcation tests ofthese results were performed using single-plex bDNA assay.
The expression levels of all 10 genes in control and 3 hTNF�-treated samples were measured. Measurements fromboth technologies reveal similar expression levels and fold-of-induction changes (Fig. 7B). The correlation (R2)between expression measurements for untreated sampleswas greater than 0.99 and for treated samples, whereas acorrelation greater than 0.94 was observed across bothpanels.
Discussion
We have developed a multiplex bDNA assay that com-bines simple and robust procedure with sensitive and repro-ducible gene expression quantiWcation directly from crudecell lysates without the need for RNA puriWcation, reversetranscription, or target ampliWcation.
Distinctive features of the multiplex bDNA assayinclude high speciWcity, accuracy, and extremely low cross-reactivity. These attributes are driven in part by the require-ment of both CE and LE to bind to the same RNAmolecule for it to be detected and in part by cooperative
Fig. 7. Time-dependent expression of pro- and anti-apoptosis genes from TNF�-treated HeLa cells. (A) Gene expression following the addition of TNF�was measured from 80,000 cell-equivalent lysates using the 10-plex apoptosis panel. GAPD measurements were obtained from 10,000 cell-equivalentlysates, and all data were acquired using the Bio-Plex system with high sensitivity calibration. (B) Expression of each gene at 3 h post-TNF� treatment wasmeasured using single-plex bDNA assay (left). Lysates from 40,000 cells were used for each gene except GAPD, where data were obtained from 10,000cell-equivalent cell lysates. Expression data obtained from multiplex bDNA assay (right) at the 3-h time point are shown for comparison. In both panels Aand B, all measurements were performed in triplicate and normalized to GAPD. Error bars represent the standard deviations of the average responses.RLU, relative luminescent units.
0100200300400500
RELB
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0 hr 1 hr 3 hr 6 hr0
200400600800
1000RELA
Net
MF
I
0200400600700
1000NFKB1
0 hr 1 hr 3 hr 6 hr
Net
MF
I
010002000300040005000
NFKB1A
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0200400600800
1000 NFKB2
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0100200300400500 CDKN1A
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0200400600800
1000 IL6
0 hr 1 hr 3 hr 6 hr
Net
MF
I
01020304050 TNFA
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0300600900
12001500 TNFAIP3
0 hr 1 hr 3 hr 6 hr
Net
MF
I
0.010.1
110
1001000
Single-plex
RELB
Net
RLU
TNFA
TNFAIP
3IL
6
CDKN1A
NFKB1
NFKB2
RELA
NFKBIA
GAPD
3 hr - TNFα3 hr + TNFα
0.010.1
110
100
10000Multiplex
RELB
Net
MF
I 1000
TNFA
TNFAIP
3IL
6
CDKN1A
NFKB1
NFKB2
RELA
NFKBIA
GAPD
3 hr - TNFα3 hr + TNFα
A
B
Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60 59
hybridization, a novel method of hybridization that lever-ages multiple weak interactions to generate a single strongand stable one. Using cooperative hybridization, a giventarget mRNA is stably captured to a solid support onlywhen the mRNA binds to multiple CEs at the same time inthe multiplexed assay, whereas nonspeciWc mRNA bindingto one CE cannot be stably captured. Thus, cooperativehybridization potentially provides two major advantagesover single probe hybridization currently employed in otherhybridization-based assays: (i) dramatic reduction in assaybackground caused by nonspeciWc cross-hybridization and(ii) better discrimination of homologous genes. By leverag-ing cooperative hybridization in the assay design, less than0.1% cross-reactivity is routinely achieved in more than adozen multiplex panels tested so far, including cytochromeP450 and ABC transporter panels where several genes withup to 96% homology are present (data not shown). Theassay speciWcity is further demonstrated by its excellentspike recovery in the presence of a complex background oftotal RNA. Because of the exceptional bDNA assay speci-Wcity, the results of mRNA quantiWcation are highly accu-rate and reliable. Finally, because known quantities of IVTRNA can be used as a reference standard, absolute num-bers of mRNA transcripts in a sample can be determined.This is in contrast to quantitative RT-PCR, where theampliWcation eYciency in a reference sample could bediVerent from that in an experimental sample. As a result, itmay be unreliable to obtain absolute quantitation ofmRNA unless competitive quantitative PCR assay is per-formed. It should be noted that application of the coopera-tive hybridization concept in other hybridization-basedassays such as microarray has the potential to signiWcantlyincrease the speciWcity and accuracy of current expressionproWling technologies.
The multiplex bDNA assay can quantify RNA tran-scripts in crude cell lysates and tissue homogenates. Thisfeature distinguishes the assay from many other existingmRNA quantiWcation technologies and makes the assayone of the few that directly measures the mRNA transcriptsfrom their native environment and thus free of any biasimposed by puriWcation procedures and enzymatic reac-tions. Eliminating the RNA puriWcation, reverse transcrip-tion, labeling, and ampliWcation steps minimizes assayvariations and leads to signiWcantly improved precision forthe overall assay. In addition, by eliminating the need forsample preparation, the multiplex bDNA assay is wellsuited for high-throughput expression analysis of largesample populations.
The multiplex bDNA assay has high precision andreproducibility. The intraplate CV is routinely less than10%, and the interplate CV is routinely less than 15% in allof our experiments where triplicate samples are run,whether the samples are total RNA or cell lysates. Becausethe assay depends on signal ampliWcation rather than targetampliWcation, the multiplex bDNA assay bypasses many ofthe variability issues faced by other technologies. In addi-tion, the unique cooperative hybridization may also
contribute to its high precision and reproducibility byenabling more consistent hybridization. The high reproduc-ibility of bDNA assays is in contrast to quantitative RT-PCR, where numerous steps have been shown to contributeto the source of variations [4]. The level of consistency inmultiplex bDNA assay data is a particularly attractive fea-ture required for quantitative measurement of gene expres-sion in siRNA knockdown, structure–activity relationship,drug dose response, and drug screening applications wherechanges in gene expression as small as 10 to 30% need to bereliably determined and diVerentiated. In addition, smalldiVerences in gene expression are relevant to the evolutionof gene regulation and to organismal function and pheno-type [25,26].
The multiplex bDNA assay currently has a detection sen-sitivity of 25,000 mRNA transcripts, which is signiWcantlygreater than that of the nuclease protection-based multiplexassay and is comparable to that of the Invader-based multi-plex assay [7,8]. There are a number of ways in which to fur-ther improve the detection sensitivity of the multiplex bDNAassay. First, the assay volume and the number of beads usedin the assay can be reduced to enhance the assay sensitivity.Reducing the assay volume to 50�l has been shown toincrease the detection sensitivity by 80% (data not shown).Second, the probe set for the mRNA of interest can beexpanded to include a larger number of LEs so that moreampliWcation molecules can bind to the mRNA. Third, a newampliWcation molecule with higher theoretical fold-of-ampli-Wcation can be used in the assay for increased signal ampliW-cation. As demonstrated in the HIV and HCV detectionassay systems, mRNA transcripts as low as 50 copies can bereliably detected in the bDNA assay [11,12].
Although the current multiplex bDNA assay simulta-neously quantiWes 10 mRNA targets in a single assay, theLuminex platform is designed for simultaneous measurementof up to 100 bead analytes and we have performed 30- and40-plex bDNA assays with the same performance character-istics (not shown). Expansion of the multiplex bDNA assaywill permit more detailed pathway analyses and enable morerobust systems biology studies. Increased multiplex capabil-ity may be desirable for some applications such as micro-array validation and follow-up biological studies.
In summary, the simplicity, sensitivity, accuracy, andhigh reproducibility of the assay, together with the multi-plex capability, make the multiplex bDNA assay a valuabletool in applications where accurate and robust quantiWca-tion of multiple mRNA targets is required, where samplesand reagents are precious and limited, and where easy andhigh-throughput sample processing is desired such as inbiomarker validation, compound screening, structure–activity relationship studies, toxicity studies, clinical trials,and clinical diagnostics.
Acknowledgments
We thank Mickey Urdea and Quan Nguyen for adviceand helpful discussions, Kate Stankis and Margaret Hirst
60 Multiplex branched DNA assay for expression proWling / M. Flagella et al. / Anal. Biochem. 352 (2006) 50–60
for graphic assistance, and Melanie Mahtani for thoughtfulreview of the manuscript. We also thank all members of theGenospectra team for their support.
[2] T.R. Golub, D.K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J.P.Mesirov, H. Coller, M.L. Loh, J.R. Downing, M.A. Caligiuri, C.D.BloomWeld, E.S. Lander, Molecular classiWcation of cancer: class dis-covery and class prediction by gene expression monitoring, Science286 (1999) 531–537.
[3] E.C. Gunther, D.J. Stone, R.W. Gerwien, P. Bento, M.P. Heyes, Predictionof clinical drug eYcacy by classiWcation of drug-induced genomic expres-sion proWles in vitro, Proc. Natl. Acad. Sci. USA 100 (2003) 9608–9613.
[4] S.A. Bustin, QuantiWcation of mRNA using real-time reverse tran-scription PCR (RT-PCR): trends and problems, J. Mol. Endocrinol.29 (2002) 23–39.
[5] S.A. Bustin, T. Nolan, Pitfalls of quantitative real-time reverse-tran-scription polymerase chain reaction, J. Biomol. Tech. 15 (2004) 155–166.
[6] C.A. White, L.A. Salamonsen, A guide to issues in microarray analysis:application to endometrial biology, Reproduction 130 (2005) 1–13.
[7] R.R. Martel, I.W. Botros, M.P. Rounseville, J.P. Hinton, R.R. Staples,D.A. Morales, J.B. Farmer, B.E. Seligmann, Multiplexed screeningassay for mRNA combining nuclease protection with luminescentarray detection, Assay Drug Dev. Technol. 1 (2002) 61–71.
[8] H. Tian, L. Cao, Y. Tan, S. Williams, L. Chen, T. Matray, A. Chenna,S. Moore, V. Hernandez, V. Xiao, M. Tang, S. Singh, MultiplexmRNA assay using electrophoretic tags for high-throughput geneexpression analysis, Nucleic Acids Res. 32 (2004) e126.
[9] M. Urdea, T. Horn, T.J. Fultz, M. Anderson, J.A. Running, S. Ham-ren, D. Ahle, C.A. Chang, Branched DNA ampliWcation multimersfor the sensitive, direct detection of human hepatitis virus, NucleicAcids Symp. Ser. 24 (1991) 197–200.
[10] J.D. Yao, M.G. Beld, L.L. Oon, C.H. Sherlock, J. Germer, S. Menting,S.Y. Se Thoe, L. Merrick, R. Ziermann, J. Surtihadi, H.J. Hnatyszyn,Multicenter evaluation of the VERSANT hepatitis B virus DNA 3.0assay, J. Clin. Microbiol. 42 (2004) 800–806.
[11] T. Elbeik, J. Surtihadi, M. Destree, J. Gorlin, M. Holodniy, S.A. Jor-tani, K. Kuramoto, V. Ng, R. Valdes Jr., A. Valsamakis, N.A. Terra-ult, Multicenter evaluation of the performance characteristics of theBayer VERSANT HCV RNA 3.0 assay (bDNA), J. Clin. Microbiol.42 (2004) 563–569.
[12] C.A. Gleaves, J. Welle, M. Campbell, T. Elbeik, V. Ng, P.E. Taylor, K.Kuramoto, S. Aceituno, E. Lewalski, B. Joppa, L. Sawyer, C. Schaper,D. McNairn, T. Quinn, Multicenter evaluation of the Bayer VER-SANT HIV-1 RNA 3.0 assay: analytical and clinical performance, J.Clin. Virol. 25 (2002) 205–216.
[13] D.P. Hartley, C.D. Klaassen, Detection of chemical-induced diVeren-tial expression of rat hepatic cytochrome P450 mRNA transcriptsusing branched DNA signal ampliWcation technology, Drug Metab.Dispos. 28 (2000) 608–616.
[14] K.S. Bramlett, S. Yao, T.P. Burris, Correlation of farnesoid X receptorcoactivator recruitment and cholesterol 7�-hydroxylase gene repres-sion by bile acids, Mol. Genet. Metab. 71 (2000) 609–615.
[15] U. Warrior, Y. Fan, C.A. David, J.A. Wilkins, E.M. McKeegan, J.L.Kofron, D.J. Burns, Application of QuantiGene nucleic acid quantiW-cation technology for high throughput screening, J. Biomol. Screen. 5(2000) 343–352.
[16] J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M.Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John,V. Kesavan, G. Lavine, R. Pandey, T. Racie, K. Rajeev, I. Röhl, I.Toudharska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S.Limmer, M. Manoharan, H. Vornlocher, Therapeutic silencing of anendogenous gene by systemic administration of modiWed siRNAs,Nature 432 (2004) 173–178.
[18] J.D. Taylor, D. Briley, Q. Nguyen, K. Long, M.A. Iannone, M.S. Li, F.Ye, A. Afshari, E. Lai, M. Wagner, J. Chen, M.P. Weiner, Flow cyto-metric platform for high-throughput single nucleotide polymorphismanalysis, BioTechniques 30 (2001) 661–669.
[19] U. Prabhakar, E. Eirikis, H.M. Davis, Simultaneous quantiWcation ofproinXammatory cytokines in human plasma using the LabMAPassay, J. Immunol. Methods 260 (2002) 207–218.
[20] S. Bushnell, J. Budde, T. Catino, J. Cole, A. Derti, R. Kelso, M.L. Col-lins, G. Molino, P. Sheridan, J. Monahan, M. Urdea, ProbeDesigner:for the design of probe sets for branched DNA (bDNA) signal ampli-Wcation assays, Bioinformatics 15 (1999) 348–355.
[21] J.A. Running, M.S. Urdea, A procedure for productive coupling ofsynthetic oligonucleotides to polystyrene microtiter wells for hybrid-ization capture, BioTechniques 8 (1990) 276–279.
[22] L. Yang, D.K. Tran, X. Wang, BADGE, beads array for the detectionof gene expression, a high-throughput diagnostic bioassay, GenomeRes. 11 (2001) 1888–1898.
[23] R. Hass, G. Lonnemann, D. Mannel, N. Topley, A. Hartmann, L.Kohler, K. Resch, M. Goppelt-Strube, Regulation of TNF-�, IL-1,and IL-6 synthesis in diVerentiating human monoblastoid leukemicU937 cells, Leuk. Res. 15 (1991) 327–339.
[24] A. Zhou, S. Scoggin, R.B. Gaynor, N.S. Williams, IdentiWcation ofNF-�B-regulated genes induced by TNF� utilizing expression proWl-ing and RNA interference, Oncogene 22 (2003) 2054–2064.
[25] H. Yan, Z. Dobbie, S.B. Gruber, S. Markowitz, K. Romans, F.M.Giardiello, K.W. Kinzler, B. Vogelstein, Small changes in expressionaVect predisposition to tumorigenesis, Nat. Genet. 30 (2002) 25–26.
[26] J.P. Townsend, D. Cavalieri, D.L. Hartl, Population genetic variationin genome-wide gene expression, Mol. Biol. Evol. 20 (2003) 955–963.
