Better, Faster, and Less Expensive Gene Expression Analysis
Using Multiplex One-Step RT-qPCR: A Case Study ofColorectal Cancer
John Repass
ARQ Genetics, LLC, 1110 Main St, Suite B, Bastrop, TX 78602

One-Step RT-qPCR Reagents

TechNote
Bulletin 6516

Abstract
In this study, a panel of biomarkers relevant to colorectal cancer research is used in the development of triplex onestep reverse transcription quantitative PCR (RT-qPCR) assays. Implementing multiplex one-step RT-qPCR can lead tosignificant savings in time and reagent costs relative to traditional singleplex RT-qPCR. We show that results obtainedusing triplex one-step RT-qPCR are equivalent to those obtained from slower and more costly singleplex analysis.
Introduction
Colorectal cancer (CRC), the third most common cancer inboth men and women, is the second leading cause of cancer
mortality in the world. As a CRC neoplasm develops andprogresses, its pattern of gene expression changes. Thus,
differentially expressed genes can be used as biomarkers toassess and monitor CRC neoplasms (Chan et al. 2008, Jubeet al. 2012, Nummela et al. 2012, Wu et al. 2012).
Clinical and translational research commonly rely on RT-qPCR
for assessment of known or putative biomarkers to monitor
disease. Such work is often limited by the large number ofsamples that need to be processed, the size of the biomarker
panel, the high cost of the RT-qPCR reagents, and the time
involved in setting up and running the experiments. One way
to overcome these limitations is to make RT-qPCR faster andless expensive. Instead of performing reverse transcription
and qPCR in two separate steps, the two reactions can be
combined into a single one-step RT-qPCR reaction. Anotherapproach is to analyze several target genes simultaneously
in multiplex reactions. For further time and cost savings,
one-step RT-qPCR and multiplexing can be combined into
multiplex one-step RT-qPCR. In this case, an RNA sample
is combined with a one-step RT-qPCR reagent and two or
more probe-based target gene PCR assays. The mix thenundergoes reverse transcription followed by multiplex qPCR ina real-time PCR instrument.

Here we demonstrate how to design and validate triplex
one-step RT-qPCR assays using a panel of biomarkers thatis important to CRC research. We show that triplex one-step
RT-qPCR results obtained with Bio-Rad’s iTaq™ Universal
Probes One-Step Kit are equivalent to results obtained fromslower and more costly singleplex analysis. This suggests thatmultiplex one-step RT-qPCR assays can be employed as atime- and cost-saving alternative to traditional qPCR protocols.
Materials and Methods
Samples

We obtained total RNA, corresponding to matched colorectal
tumor tissue and adjacent normal tissue, from two patients
through a commercial source (BioChain Institute, Inc.).
The samples were from (1) a 68-year-old male with poorlydifferentiated adenocarcinoma and (2) a 62-year-old female
with moderately differentiated adenocarcinoma.
Target Genes

Four target genes that exhibit differential expression in CRC
samples were assessed (TGFBI, COL1A1, HMGB1, and
COL3A1). Amplification primers and probes were designed
using Primer3Plus (www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and synthesized by Integrated DNA
Technologies, Inc. (IDT). GAPDH primers and probe werefrom Biosearch Technologies, Inc. Table 1 lists the primer andprobe sequences.

Better, Faster, and Less Expensive Gene Expression Analysis Using Multiplex One-Step RT-qPCR: A Case Study of Colorectal Cancer

Table 1. Primer and probe information.
GeneTriplex Primer
Set NameTGFBI
1TGFBI F		TGFBI R		TGFBI P

Sequence(5'>3')CCACAGCCATTGACCTTTTCATCAACCGCTCACTTCCAGAGA
FAM-ACAAGCCGGCCTCGGCAATCA

COL1A11COL1A1 F CAGACAAGCAACCCAAACTGAA		COL1A1 R TGAGAGATGAATGCAAAGGAAAAA		COL1A1 P TET-CCCTCAAAAGCCAAAAAATGGGAGACAATTHMGB12HMGB1 F TGCTCTGAGTATCGCCCAAA		HMGB1 R TCCACATCTCTCCCAGTTTCTTC		HMGB1 P FAM-ACATCCTGGCCTGTCCATTGGTGA
COL3A1 2COL3A1 F GGGAATGGAGCAAAACAGTCTT		COL3A1 R CCAACGTCCACACCAAATTCT		COL3A1 P TET-TCGAACACGCAAGGCTGTGAGACTACCGAPDH 1 and 2 GAPDH F CAGCCTCAAGATCATCAGCA		GAPDH R TGTGGTCATGAGTCCTTCCA		GAPDH P Quasar 705–CTGCTTAGCACCCCTGGCCA
F, forward primer; P, probe; R, reverse primer.

Triplex Reactions

We developed two sets of triplex assays. Set 1 contains
TGFBI, COL1A1, and GAPDH. Set 2 contains HMGB1, COL3A1,and GAPDH. GAPDH serves as an internal reference gene
control in both triplex assays.
One-Step RT-qPCR

One-step RT-qPCR was performed using the iTaq UniversalProbes One-Step Kit as directed by the manufacturer on
a CFX96™ Real-Time PCR Detection System (Bio-Rad). For
all target genes, primers and probes were used at 250 nM
final concentrations. For the GAPDH reference gene,primers were used at 100 nM and the probe at 250 nM final
concentrations. The GAPDH assay included a lesser amountof primers because GAPDH is highly expressed; lowering
the concentration of GAPDH primers prevents GAPDH
amplification from depleting reaction components. Cyclingconditions were 10 min at 50°C (reverse transcription),
3 min at 95°C followed by 40 cycles of 10 sec at 95°C, and1 min at 60°C.
Triplex Assay Validation

To validate the triplex assays, triplex and singleplex one-step
RT-qPCR performance was compared using tenfold serial
dilutions of HeLa RNA (100 ng–10 pg). The experiment wasrepeated three times independently.
Analysis of CRC Samples

Each patient RNA sample (1 ng) was analyzed by one-step
RT-qPCR in both triplex and singleplex. Each sample wasanalyzed four times independently.
Results and Discussion
Triplex Assay Validation

We chose to analyze a panel of four biomarkers that arereported to be overexpressed in CRC. COL1A1, COL3A1,and TGFBI act in the extracellular matrix receptor interaction
pathway (Chan et al. 2008, Nummela et al. 2012, Wu et al.
2012), whereas HMGB1 has potential roles in tumor cell
survival and metastasis (Jube et al. 2012).
© 2014 Bio-Rad Laboratories, Inc.

We developed two sets of triplex assays. Each set contains
two CRC biomarkers and the GAPDH reference gene thatserves as an internal normalization control (Table 1). To validate
the triplex assays in one-step RT-qPCR, we compared triplex
performance with singleplex performance using serially diluted
HeLa RNA. We find that the triplex assays and singleplex
assays give equivalent performance. At all input RNA levels,
the quantification cycle (Cq) values of the triplex and singleplex
reactions are virtually indistinguishable (Figure 1) and the qPCR
efficiency and R2 values are statistically the same (Table 2).
These data indicate that triplex assays can be performed
instead of singleplex assays without a loss in data quality.
Analysis of Samples from Patients with CRC

To confirm that triplex and singleplex assays yield equivalent
results we analyzed RNA samples from two patients withCRC. We obtained two RNA samples from each patient: one
sample from a malignant colon region and the other sample
from an adjacent colon region that was not cancerous. We
then quantified the expression of each biomarker using triplex
and singleplex one-step RT-qPCR, normalized biomarker
expression with GAPDH, and determined whether eachbiomarker exhibits differential expression in cancerous
tissue. Our results show that, in the two patients analyzed,
TGFB1, COL1A1, and COL3A1 are upregulated in cancerous
tissue while HMGB1 is not (Figure 2). Importantly, the triplex
and singleplex assays produce results that are statisticallyequivalent. These findings confirm that our triplex assays
can substitute for singleplex assays without compromising
the quality of results. This implies that in future work, patient
samples can be reliably analyzed by triplex one-step RT-qPCR.
Benefits of Multiplex One-Step RT-qPCR

Our work demonstrates that properly validated one-stepRT-qPCR triplex reactions using Bio-Rad’s iTaq Universal
Probes One-Step Kit generate results that are equivalent tosingleplex reactions. Implementing multiplex one-step
RT-qPCR can lead to significant savings in time and reagent
costs relative to traditional singleplex RT-qPCR. Multiplex
one-step RT-qPCR can also lead to improved data quality
since researchers need to perform only one reaction using asingle reagent. This saves time and can lead to better results
because there is no chance of manipulation error between
the reverse transcription and qPCR steps. Multiplex analysis
also reduces sample utilization because fewer reactions need
to be processed. Lastly, because each multiplex reaction
can be internally controlled, with GAPDH in our example,
there will be less variability in experiments involving relative
gene expression.
Conclusions
Multiplex one-step RT-qPCR provides an excellent alternative
to traditional RT-qPCR protocols, especially when time,sample, and cost are limited.

Bulletin 6516Better, Faster, and Less Expensive Gene Expression Analysis Using Multiplex One-Step RT-qPCR: A Case Study of Colorectal Cancer

HMGB1-FAM

104

COL3A1-TET

104

103

102

103

10201020		Cycles

3040103

10201020		Cycles

TGFBI-FAM

104304001020		Cycles

COL1A1-TET

104

102

103

10201020		Cycles

304040Singleplex
Triplex

RFU

10330

GAPDH–Quasar 705

104

Singleplex
Triplex

RFU

Singleplex
Triplex

RFU

Singleplex
Triplex

RFU

Singleplex
TriplexRFU

RFU

Singleplex
TriplexGAPDH–Quasar 705

104

103

10201020		Cycles

304001020		Cycles

3040Fig. 1. Validation of triplex assays. Amplification plots of tenfold serial RNA dilutions are shown (100 ng–10 pg). For all targets, the Cq values for the singleplex
and triplex reactions are virtually indistinguishable, indicating that triplex reactions perform as well as singleplex reactions. RFU, relative fluorescence units.
Table 2. Comparison of singleplex and triplex one-step RT-qPCR reactions.*
R2

Efficiency, %Gene

SetSingleplex

SDTriplex

SDSingleplex

SDTriplex

SD

TGFBI (FAM)

1

99.1

4.12

100.4

1.44

0.999

0.00058

1.000

0.00000
0.00100

COL1A1 (TET)1100.7

4.87

104.8

3.70

0.998

0.00058

0.999

GAPDH (Quasar 705)

197.2

1.88

99.1

1.05

0.999

0.00058

0.9990.00058

HMGB1 (FAM)

299.0

1.74

100.1

0.90

1.000

0.00000

1.000

0.00000

COL3A1 (TET)

299.1

0.40

101.1

0.83

1.000

0.00000

1.0000.00000

GAPDH (Quasar 705)

297.2

1.91

99.0

1.00

1.000

0.00000

1.0000.00000* Efficiency and R2 values were obtained from standard curve assays. Standard curves were generated with 4-log serial dilutions of RNA (100 ng–10 pg). SD is thestandard deviation from three independent standard curve analyses.

Tumor/normal fold

Patient 1

Patient 26325164834221100.5
Singleplex

TriplexTGFBI

Singleplex

Triplex

COL1A1

Singleplex

Triplex

COL3A1

Singleplex

Triplex

HMGB1

Singleplex

TriplexTGFBI

Singleplex

Triplex

COL1A1

Singleplex

Triplex

COL3A1

Singleplex

Triplex

HMGB1

Fig. 2. Differential biomarker expression in matched pairs of cancerous and adjacent normal tissue RNA samples. Gene expression levels in RNA samples
from two patients with CRC were determined using triplex and singleplex one-step RT-qPCR. Biomarker expression was normalized using the GAPDH internalreference gene control. The ratio of biomarker expression in cancerous tissue relative to adjacent normal tissue is shown for each patient. The experiment wasrepeated four times; error bars represent the 95% confidence interval.

© 2014 Bio-Rad Laboratories, Inc.

Bulletin 6516

Better, Faster, and Less Expensive Gene Expression Analysis Using Multiplex One-Step RT-qPCR: A Case Study of Colorectal Cancer

References
Chan SK et al. (2008). Meta-analysis of colorectal cancer gene expression
profiling studies identifies consistently reported candidate biomarkers. Cancer
Epidemiol Biomarkers Prev 17, 543–552.
Jube S et al. (2012). Cancer cell secretion of the DAMP protein HMGB1
supports progression in malignant mesothelioma. Cancer Res 72, 3290–3301.
Nummela P et al. (2012). Transforming growth factor beta-induced (TGFBI) is ananti-adhesive protein regulating the invasive growth of melanoma cells. Am J
Pathol 180, 1663–1674.
Wu Y et al. (2012). Transcriptome profiling of the cancer, adjacent nontumor and distant normal tissues from a colorectal cancer patient by deep
sequencing. PLoS One 7, e41001.

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Numbers 6,767,512 and 7,074,367.
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